Microfluidic system and method with focused energy apparatus

ABSTRACT

A method of inseminating an animal including flowing a stream of a population of sperm cells through a channel, differentiating the sperm cells into two subpopulations of X-chromosome containing sperm cells and Y-chromosome containing sperm cells, selecting a desired subpopulation, ablating an undesired subpopulation, and collecting both the subpopulations of sperm cells including the desired subpopulation and the ablated undesired subpopulation together, wherein the collected population of sperm cells is used to fertilize an egg.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/496,469 filed Oct. 7, 2021 and of U.S. patent application Ser. No.17/496,614 filed Oct. 7, 2021, both of which are continuations of U.S.patent application Ser. No. 16/279,430, filed Feb. 19, 2019, which is acontinuation-in-part of U.S. patent application Ser. No. 15/387,034,filed Dec. 21, 2016, now abandoned, which is a continuation of U.S.patent application Ser. No. 15/033,001, filed Aug. 24, 2016, now U.S.Pat. No. 10,928,298, which is a 371 of International ApplicationPCT/IB2014/001425, filed Jun. 18, 2014, which claims priority to U.S.Provisional Patent Application No. 61/897,743, filed Oct. 30, 2013, thespecifications of which are herein incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to a microfluidic system with aninterrogation apparatus that detects and interrogates objects in asample fluid mixture of a microfluidic chip, and a focused energyapparatus that performs an action that affects the objects. In oneembodiment, the interrogation apparatus interrogates the objects todetermine their identity, and the focused energy apparatus is anapparatus that acts on target objects. In one embodiment, the focusedenergy apparatus is used to damage, kill, alter, disable, or destroy thetargeted objects.

BACKGROUND OF THE INVENTION

In the separation of various particles or cellular materials—forexample, the separation of sperm into viable and motile sperm fromnon-viable or non-motile sperm, or separation by gender—the process isoften a time-consuming task, with severe volume restrictions. Thus,current separation techniques cannot, for example, produce the desiredyield, or process volumes of cellular materials in a timely fashion.

Photo-damaging laser systems have utilized lasers to photodamage or killundesired cellular objects. However, the prior art has required flowcytometers using nozzles, to interrogate and arrange the individualobjects in droplet flow, and to attempt to separate and photodamage theobjects as they fall into various containers—which has been difficult toachieve.

Thus, there exists a present need for a method and apparatus whichidentifies and discriminates between target objects, is continuous, hashigh throughput, is time and cost-effective, and causes negligible orminimal damage to the various target objects. In addition, such anapparatus and method should have further applicability to otherbiological and medical areas, not just in sperm discrimination; but inthe discrimination of blood and other cellular materials, includingviral, cell organelle, globular structures, colloidal suspensions, andother biological materials.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to a microfluidic system with aninterrogation apparatus that detects and interrogates objects in asample fluid mixture of a microfluidic chip, and a focused energyapparatus that performs an action that affects the objects. In oneembodiment, the interrogation apparatus interrogates the objects todetermine their identity, and the focused energy apparatus is anapparatus that acts on target objects. In one embodiment, the focusedenergy apparatus is used to damage, kill, alter, disable, or destroy thetargeted objects.

In one embodiment, an apparatus that identifies objects includes: amicrofluidic chip in which are disposed a plurality of channels;including; a main fluid channel into which a sample fluid mixture ofobjects to be identified is introduced; a plurality of sheath fluidchannels into which sheath fluids are introduced, the sheath fluidswhich orient the objects in the main fluid channel in a predetermineddirection while still maintaining laminar flow in the main fluidchannel; an interrogation apparatus which detects and interrogates theoriented objects in the main fluid channel; and a focused energyapparatus which performs an action on the objects.

In one embodiment, the interrogation apparatus detects and interrogatesthe objects to determine information about the objects.

In one embodiment, the information about the objects determines whetherthe objects are targeted by the focused energy apparatus.

In one embodiment, the action of the focused energy apparatus acts onthe targeted objects or a region surrounding the targeted objects.

In one embodiment, the action on the targeted objects is to damage,disable, alter, kill or destroy the targeted objects.

In one embodiment, the apparatus further includes at least one outputchannel leading from the main fluid channel, the at least one outputchannel which removes the objects from the microfluidic chip.

In one embodiment, the at least one output channel removes both targetedand non-targeted objects from the microfluidic chip.

In one embodiment, the apparatus further includes a plurality of sideoutput channels leading from the main fluid channel, the plurality ofside output channels disposed on either side of the at least one outputchannel, the plurality of side output channels which remove the sheathfluids from the microfluidic chip.

In one embodiment, the plurality of sheath fluid channels includes: afirst plurality of sheath fluid channels which intersect the main fluidchannel at a first intersection, such that the sheath fluids compressthe sample fluid mixture on at least two sides, such that the samplefluid mixture becomes a relatively smaller, narrower stream, bounded bythe sheath fluids while maintaining laminar flow in the main fluidchannel.

In one embodiment, the plurality of sheath fluid channels furtherincludes: a second plurality of sheath fluid channels which intersectthe main fluid channel at a second intersection downstream from thefirst intersection, such that the sheath fluids from the secondplurality of sheath fluid channels compress the sample fluid mixture inone of the at least two sides, or in two sides opposite from the atleast two sides, such that the sample fluid mixture is furthercompressed while still maintaining laminar flow in the main fluidchannel.

In one embodiment, when the second set of sheath fluid channelscompresses the sample fluid mixture from the at least two sides, theplurality of sheath fluid channels further comprises: a third sheathfluid channel disposed vertical to the main fluid channel at a thirdintersection, and disposed downstream from the second intersection, thesheath fluid from the third sheath fluid channel which furthercompresses the sample fluid while still maintaining laminar flow in themain fluid channel.

In one embodiment, the plurality of sheath fluid channelshydrodynamically focuses the objects such that the objects are orientedin a predetermined direction and disposed in a restricted core volume asthe objects flow through the main fluid channel.

In one embodiment, the apparatus further includes an action chamber inwhich the interrogation apparatus interrogates the hydrodynamicallyfocused objects in the sample fluid mixture, the action chamber disposedin the microfluidic chip downstream from at least one of the secondintersection or the third intersection.

In one embodiment, the interrogation apparatus includes; a light sourcethat emits a light beam into the action chamber, to illuminate andexcite the objects in the sample fluid mixture.

In one embodiment, the light beam excites fluorescence in the objectssuch that the targeted objects are distinguished from the non-targetedobjects.

In one embodiment, the light source is a laser.

In one embodiment, the apparatus further includes an optical signaldetector which detects the light beam and converts it into an electronicsignal; and a controller, which analyzes the electronic signal todetermine whether the objects are to be targeted or non-targeted.

In one embodiment, the focused energy apparatus is a laser.

In one embodiment, the microfluidic chip contains one or more structurallayers or planes.

In one embodiment, the main fluid channel is disposed in a differentstructural layer or plane from the plurality of sheath channels.

In one embodiment, the at least one of the sample input channel and theplurality of sheath channels are disposed in-between the structurallayers or the planes of the microfluidic chip.

In one embodiment, the first plurality of sheath channels is disposed ina different structural layer or plane from the second plurality ofsheath channels.

In one embodiment, the action chamber includes a first opening cutthrough at least one of the structural layers or the planes in themicrofluidic chip, the first opening which is configured to receive afirst transparent covering.

In one embodiment, the action chamber includes a second opening cutthrough the at least one of the structural layers or the planes on anopposite side of the microfluidic chip from the first opening, thesecond opening which is configured to receive a second transparentcovering.

In one embodiment, the microfluidic chip contains at least onefunctional layer which includes the plurality of sheath fluid channelsand the main fluid channel, and a top layer that contains holes toaccess the at least one functional layer.

In one embodiment, a size of one of the second plurality of sheath fluidchannels is different from another of the second plurality of sheathchannels.

In one embodiment, the size of the second plurality of sheath channelsis different from a size of the first plurality of sheath channels.

In one embodiment, the apparatus further includes a first outputdisposed at an end of the at least one output channel.

In one embodiment, the apparatus further includes a plurality of outputsdisposed at an end of each of the plurality of side output channels.

In one embodiment, the apparatus further includes at least one notchdisposed in the microfluidic chip, the at least one notch providedbetween outputs.

In one embodiment, a size of the plurality of side output channelsincreases from a size of the main fluid channel.

In one embodiment, the main fluid channel tapers at an entry point intothe first intersection in the microfluidic chip.

In one embodiment, the main fluid channel tapers into the actionchamber.

In one embodiment, the second plurality of sheath channels tapers beforejoining the main fluid channel.

In one embodiment, the second plurality of sheath channels includes atleast a first vertical portion which joins the main fluid channel fromapproximately a right angle above the main fluid channel.

In one embodiment, the second plurality of sheath channels includes asecond vertical portion which joins the main fluid channel fromapproximately a right angle below the main fluid channel.

In one embodiment, the internal ramps are disposed in at least the mainfluid channel prior to the first intersection.

In one embodiment, the internal ramps are disposed in the main fluidchannel prior to the second intersection.

In one embodiment, the internal ramps are disposed in at least one ofthe second plurality of sheath channels.

In one embodiment, the objects are cells.

In one embodiment, the cells to be acted upon by the focused energyapparatus include at least one of viable or motile sperm from non-viableor non-motile sperm or sperm discriminated by gender or other sexdiscrimination variations.

In one embodiment, the cells to be acted upon by the focused energyapparatus include: stem cells discriminated from cells in a population;one or more labeled cells discriminated from unlabeled cells; cellsdiscriminated by desirable or undesirable traits; cells discriminatedbased on surface markers; cells discriminated based on membraneintegrity or viability; cells having genes which are discriminated innuclear DNA according to a specified characteristic; cells discriminatedbased on potential or predicted reproductive status; cells discriminatedbased on an ability to survive freezing; cells discriminated fromcontaminants or debris; healthy cells discriminated from damaged cells;red blood cells discriminated from white blood cells and platelets in aplasma mixture, or any cells discriminated from any other cellularobjects into corresponding fractions.

In one embodiment, the laser is one of a 349 or 355 nm pulsed laser.

In one embodiment, the laser is a pulsed Q-switch laser able to deliver15 ns or shorter energy pulses to the objects at a rate of over 1,000pulses per second.

In one embodiment, the laser is a 532 nm laser.

In one embodiment, the pulsed Q-switch laser preferably delivers 10 nsenergy pulses to the objects at a rate of over 200,000 pulses persecond.

In one embodiment, the focused energy apparatus acts upon the objects apredetermined amount of time after the interrogation of the objects.

In one embodiment, the focused energy apparatus acts upon the objectsprior to interrogation of the objects by the light source.

In one embodiment, the focused energy apparatus acts upon the objectswhen the objects leave the at least one output prior to being collectedin a container.

In one embodiment, the apparatus further includes a container thatcollects both the targeted and the non-targeted objects.

In one embodiment, the apparatus further includes a pumping apparatusthat pumps at least one of the sample fluid mixture or the plurality ofsheath fluids into the microfluidic chip.

In one embodiment, the pumping apparatus pumps the at least one of thesample fluid mixture or the plurality of sheath fluids into themicrofluidic chip using external tubing.

In one embodiment, the apparatus further includes: at least one externalreservoir which holds at least one of the sample fluid mixture or theplurality of sheath fluids.

In one embodiment, the apparatus further includes: a microfluidic chipholder on which the microfluidic chip is mounted, the microfluidic chipholder which includes openings through which the external tubingaccesses the microfluidic chip from the at least one external reservoir.

In one embodiment, the apparatus further includes: a controller whichcontrols the pumping of the at least one of the sample fluid mixture orthe plurality of sheath fluids into the microfluidic chip.

In one embodiment, the apparatus further includes a plurality ofmicrofluidic chips disposed in parallel, the plurality of microfluidicchips containing a plurality of sample fluid mixtures; wherein a singleinterrogation apparatus is used for each of the plurality ofmicrofluidic chips.

In one embodiment, a computer system identifies objects, including: atleast one memory which contains at least one program which includes thesteps of: controlling a flow of a sample fluid mixture containingobjects to be identified, through a main fluid channel of a microfluidicchip; controlling an introduction of a plurality of sheath fluidchannels into the microfluidic chip, the plurality of sheath fluidswhich orient the objects in the main fluid channel in a predetermineddirection while still maintaining laminar flow in the main fluidchannel; and analyzing an interrogation of the oriented objects in themain fluid channel using an interrogation apparatus; and controlling anaction on the objects using a focused energy apparatus; and a processorwhich executes the program.

In one embodiment, a non-transitory computer-readable medium containinginstructions to identify objects includes: controlling a flow of asample fluid mixture containing objects to be identified, through a mainfluid channel of a microfluidic chip; controlling an introduction of aplurality of sheath fluid channels into the microfluidic chip, theplurality of sheath fluids which orient the objects in the main fluidchannel in a predetermined direction while still maintaining laminarflow in the main fluid channel; analyzing an interrogation of theoriented objects in the main fluid channel using an interrogationapparatus; and controlling an action on the objects using a focusedenergy apparatus.

In one embodiment, an apparatus that identifies objects includes: amicrofluidic chip in which are disposed a plurality of channels,including: a main fluid channel into which a sample fluid mixture ofobjects to be identified is introduced; and a plurality of sheath flowchannels which perform at least a three-step hydrodynamic focusingprocess on the objects, such that the objects are oriented in apredetermined direction as the objects flow through the main fluidchannel.

In one embodiment, the plurality of sheath fluid channels includes: afirst plurality of sheath fluid channels which intersect the main fluidchannel at a first intersection to accomplish a first step of the atleast three hydrodynamic focusing steps, such that the sheath fluidscompress the sample fluid mixture on at least two sides, such that thesample fluid mixture becomes a relatively smaller, narrower stream,bounded by the sheath fluids while maintaining laminar flow in the mainfluid channel.

In one embodiment, the plurality of sheath fluid channels furtherincludes: a second plurality of sheath fluid channels which intersectthe main fluid channel at a second intersection downstream from thefirst intersection to accomplish a second step of the at least threehydrodynamic focusing steps, such that the sheath fluids from the secondplurality of sheath fluid channels further compress the sample fluidmixture in the at least two sides, such that the sample fluid mixture isfurther compressed while still maintaining laminar flow in the mainfluid channel.

In one embodiment, the plurality of sheath fluids further includes: athird sheath fluid channel disposed vertical to the main fluid channelat a third intersection, and disposed downstream from the secondintersection to accomplish a third step of said at least threehydrodynamic focusing steps, the sheath fluid from the third sheathfluid channel which compresses the sample fluid while still maintaininglaminar flow in the main fluid channel.

In one embodiment, the apparatus further includes: an interrogationapparatus that detects and interrogates the oriented objects in the mainfluid channel; and a focused energy apparatus that performs an action onthe objects.

In one embodiment, a method of identifying objects flowing in a samplefluid mixture includes: flowing a sample fluid mixture containingobjects to be identified, through a main fluid channel of a microfluidicchip; introducing a plurality of sheath fluid channels into themicrofluidic chip, the plurality of sheath fluids which orient theobjects in the main fluid channel in a predetermined direction whilestill maintaining laminar flow in the main fluid channel; interrogatingthe oriented objects in the main fluid channel using an interrogationapparatus; and using a focused energy apparatus on the objects.

Thus has been outlined, some features consistent with the presentinvention so that the detailed description thereof that follows may bebetter understood, and so that the present contribution to the art maybe better appreciated. There are, of course, additional featuresconsistent with the present invention that will be described below andwhich will form the subject matter of the claims appended hereto.

In this respect, before explaining at least one embodiment consistentwith the present invention in detail, it is to be understood that theinvention is not limited in its application to the details ofconstruction and the arrangements of the objects set forth in thefollowing description or illustrated in the drawings. Methods andapparatuses consistent with the present invention are capable of otherembodiments and of being practiced and carried out in various ways.Also, it is to be understood that the phraseology and terminologyemployed herein, as well as the abstract included below, are for thepurpose of description and should not be regarded as limiting.

As such, those skilled in the art will appreciate that the conceptionupon which this disclosure is based may readily be utilized as a basisfor the designing of other structures, methods, and systems for carryingout the several purposes of the present invention. It is important,therefore, that the claims be regarded as including such equivalentconstructions insofar as they do not depart from the spirit and scope ofthe methods and apparatuses consistent with the present invention.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

The objects, features, and advantages of the present invention will bemore readily appreciated upon reference to the following disclosure whenconsidered in conjunction with the accompanying drawings, in which:

FIG. 1A shows an exploded perspective view of an illustrative embodimentof a single layer microfluidic chip with a top “blank” layer, accordingto one embodiment consistent with the present invention.

FIG. 1B (a) shows an exploded perspective view of an illustrativeembodiment of a two-layer microfluidic chip, with the functional layerson the top of the bottom layer, and on the underside of the top layer,according to yet another embodiment consistent with the presentinvention.

FIG. 1B (b) shows a top view of the bottom layer and the underside ofthe top layer of the embodiment shown in FIG. 1B (a).

FIG. 1C (a) shows an exploded perspective view of an illustrativeembodiment of a three-layer microfluidic chip with three functionallayers, the top and middle layers having the functional portions on theunderside of the layers, according to yet another embodiment consistentwith the present invention.

FIG. 1C (b) shows the three-layer illustrative embodiment of FIG. 1C(a), from the opposite perspective, with the layers flipped over,showing the underside, functional layers of the top and middle layers.

FIG. 1D shows an exploded perspective view of an illustrative embodimentof a four-layer microfluidic chip with the top layer being a “blank”layer, according to yet another embodiment consistent with the presentinvention.

FIG. 2 shows a top view of an illustrative embodiment of a microfluidicchip, according to one embodiment consistent with the present invention.

FIG. 3A shows a perspective view of the sample, and sheath or bufferchannels in a two (functional) layer microfluidic chip, according to oneembodiment consistent with the present invention.

FIG. 3B shows a perspective view of the sample, and sheath or bufferchannels in a single layer microfluidic chip, according to anotherembodiment consistent with the present invention.

FIG. 4A shows a perspective view of the sample channel, with a taper andinternal ramp, entering the intersection of the first hydrodynamicfocusing region according to one embodiment consistent with the presentinvention.

FIG. 4B shows a perspective view of the main channel, with a taper andinternal ramp, entering the second hydrodynamic focusing region,according to one embodiment consistent with the present invention.

FIG. 4C shows a ramp feature in the sample channel prior to the actionchamber, according to one embodiment consistent with the presentinvention.

FIG. 5 shows a cross-sectional internal view of an illustrativeinterrogation by a light source, of objects flowing in a fluid mixturethrough the main channel of the microfluidic chip system of FIG. 1A,according to one embodiment consistent with the present invention.

FIG. 6A shows a slanted, schematic side view of the flow of objects inthe main channel of a microfluidic chip system, from interrogation todiscrimination, with activation of the focused energy device afterinterrogation, according to one embodiment consistent with the presentinvention.

FIG. 6B shows a slanted, schematic side view of the flow of objects inthe main channel of a microfluidic chip system, from interrogation todiscrimination, with activation of the focused energy device at outputchannel exit, according to another embodiment consistent with thepresent invention.

FIG. 6C shows a slanted, schematic side view of the flow of objects inthe main channel of a microfluidic chip system, from interrogation todiscrimination, with activation of the focused energy device on adisconnected droplet between output channel exit and collection,according to another embodiment consistent with the present invention.

FIG. 6D shows a slanted, schematic side view of the flow of objects inthe main channel of a microfluidic chip system, from interrogation todiscrimination, with activation of the focused energy device prior tointerrogation, according to another embodiment consistent with thepresent invention.

FIG. 7 shows a perspective view of the shear force gradient across themicrofluidic main channel, according to one embodiment consistent withthe present invention.

FIG. 8A shows a cross-sectional view of the main channel of amicrofluidic chip, after the first-step hydrodynamic focusing, with thesample fluid compressed on the sides of the main channel, and theobjects offset from the central portion of the main channel.

FIG. 8B shows a cross-sectional view of the main channel of amicrofluidic chip, after second-step hydrodynamic focusing, with thesample fluid compressed from above and below the main channel, such thatthe objects are substantially centrally located in the main channel.

FIG. 9 shows a cross-section of the main channel of the microfluidicchip, with an object in the center thereof, according to one embodimentconsistent with the present invention.

FIG. 10 is a diagram showing a flow velocity profile across the mainchannel of a microfluidic chip, according to one embodiment consistentwith the present invention.

FIG. 11 shows a cross-section of the main channel of a microfluidicchip, with an object offset from the center, according to one embodimentconsistent with the present invention.

FIG. 12A shows the second hydrodynamic focusing step, where sheath orbuffer channels parallel the sample channel of a microfluidic chip, fromabove and below, and enter the sample channel from vertical directions,according to one embodiment consistent with the present invention,

FIG. 12B shows the second hydrodynamic focusing step of FIG. 12A, withone sheath or buffer channel of a microfluidic chip, being smaller thanthe other channel, according to another embodiment consistent with thepresent invention.

FIG. 13A shows a histogram of sperm cells in a center of the mainchannel of a microfluidic chip, according to one embodiment consistentwith the present invention.

FIG. 13B shows a histogram of sperm cells offset from a center of themain channel of a microfluidic chip, according to one embodimentconsistent with the present invention.

FIG. 14 shows a perspective, internal and oblique view of objectsflowing through the microfluidic chip, and an illustrative operation oftwo-step hydrodynamic focusing, with a single sheath or buffer fluidsentering from an upper portion of the main channel, according to oneembodiment consistent with the present invention.

FIG. 15 shows a perspective view of the microfluidic chip, and anillustrative operation of two-step hydrodynamic focusing, with sheath orbuffer fluid channels entering vertically from both an upper and a lowerportion of the sample channel, according to one embodiment consistentwith the present invention.

FIG. 16 shows a slanted, side schematic view of the microfluidic chipsystem with interrogation and discrimination apparatus, andimplementation of the beam optics from opposite sides of the samplechannel, with the collection optics shared with the focused energydevice optics, according to one embodiment consistent with the presentinvention.

FIG. 17 shows a slanted, side schematic view of the microfluidic chipsystem with interrogation and discrimination apparatus, andimplementation of the beam optics from a same side of the samplechannel, with the interrogation and focused energy beams combinedthrough the same, or separate optics, according to one embodimentconsistent with the present invention.

FIG. 18 shows a schematic view of multiple microfluidic chip systemsdisposed in parallel, using a single interrogation apparatus, accordingto one embodiment consistent with the present invention.

FIG. 19 shows a schematic view of the flow control network of themicrofluidic chip system, with external individual reservoirs for sampleand sheath or buffer fluids, according to one embodiment consistent withthe present invention.

FIG. 20 shows a schematic view of the flow control network of themicrofluidic chip system, with an external single sheath or bufferreservoir and sample reservoir, according to one embodiment consistentwith the present invention.

FIG. 21 shows a schematic view of a pressure-regulated network of themicrofluidic chip system with a single sheath or buffer reservoir,according to one embodiment consistent with the present invention.

FIGS. 22A and 223 show the front and back, respectively, of amicrofluidic chip holder having three ports for fluids to a functionalmultilayer microfluidic chip, according to one embodiment consistentwith the present invention,

FIGS. 23A and 233 show the front and back, respectively, of amicrofluidic chip holder having four ports for fluids to a single layermicrofluidic chip, according to one embodiment consistent with thepresent invention.

FIG. 24 shows the total DNA comparison between a female sperm cell and amale sperm cell.

FIG. 25 shows staining of DNA in cells to determine the intensity ofemitted light which correlates with the amount of DNA in a cell, wherein3.8% more light emitted correlates to 3.8% more DNA in female spermcells.

FIG. 26 shows the excitation wavelength and the emission wavelength forcells stained with Hoescht 33342.

FIG. 27 shows a multiple station setup to analyze cells with a vanguardlaser.

FIG. 28 shows beam splitting occurring for multiple stations.

FIG. 29 shows that the Stoke shift and optical filter create a peak at450 nm wavelength—therefore setting our detection wavelength.

FIG. 30A shows the knife blade shape, which requires tight focusing of acell stream.

FIG. 30B shows that a kill pulse is triggered based on a timer when acell is detected.

DETAILED DESCRIPTION OF THE INVENTION

Before turning to the figures, which illustrate the illustrativeembodiments in detail, it should be understood that the presentdisclosure is not limited to the details or methodology set forth in thedescription or illustrated in the figures. It should also be understoodthat the terminology is for the purpose of description only and shouldnot be regarded as limiting. An effort has been made to use the same orlike reference numbers throughout the drawings to refer to the same orlike parts.

The present invention relates to a microfluidic chip with aninterrogation apparatus that detects and interrogates objects in asample fluid mixture, and a focused energy apparatus that performs anaction on the objects or a region around the objects. In one embodiment,the interrogation apparatus interrogates the objects to identify theobjects and to determine whether the objects should be targeted by thefocused energy apparatus. In one embodiment, the targeted objects areunwanted targeted objects.

In one embodiment, the focused energy apparatus is a discriminationapparatus that discriminates between targeted and non-targeted objectsby damaging, killing, altering; disabling, or destroying the targetedobjects. The present invention is conducted in a flowing, continuousfluid stream within the microfluidic network, where objects are subjectto hydrodynamic focusing, positioning, and orientation, and non-targetedobjects are allowed to flow through the microfluidic chip undisturbed,and targeted objects may be acted upon, including photodamaged, killed,altered, disabled, or destroyed, by a focused energy apparatus.

Applications F: The various embodiments of the present invention providefor the selection of objects in a fluid mixture, such as, for example:selecting viable or motile sperm from non-viable or non-motile sperm;selecting sperm by gender, and other sex selection variations; selectingstems cells from cells in a population; selecting one or more labeledcells from un-labeled cells distinguishing desirable/undesirable traits;selecting cells for desirable characteristics; selecting genes innuclear DNA in cells, according to a specified characteristic; selectingcells based on surface markers; selecting cells based on membraneintegrity (viability), potential or predicted reproductive status(fertility), ability to survive freezing, etc.; selecting cells fromcontaminants or debris; selecting healthy cells from damaged cells(i.e., cancerous cells) (as in bone marrow extractions); red blood cellsfrom white blood cells and platelets in a plasma mixture; and selectingany cells from any other cellular objects, into corresponding fractions;selecting damaged cells, or contaminants or debris, or any otherbiological materials that are desired to discriminated. The objects maybe cells or beads treated or coated with linker molecules or embeddedwith a fluorescent or luminescent label molecule(s). The objects mayhave a variety of physical or chemical attributes, such as size, shape,materials, texture, etc.

In one embodiment, a heterogeneous population of objects may bemeasured, with each object being examined for different quantities orregimes in similar quantities (e.g., multiplexed measurements), or theobjects may be examined and distinguished based on a label (e.g.,fluorescent), image (due to size, shape, different absorption,scattering, fluorescence, luminescence characteristics, fluorescence orluminescence emission profiles, fluorescent or luminescent decaylifetime), and/or particle position, etc.

In addition, the subject matter of the present disclosure is alsosuitable for other medical applications as well. For example, thevarious laminar flows discussed below may be utilized as part of akidney dialysis process, in which whole blood is cleansed of wasteproducts and returned to the patient. Further, the various embodimentsof the present disclosure may have further applicability to otherbiological or medical areas, such as for selection of cells, viruses,bacteria, cellular organelles or subparts, globular structures,colloidal suspensions, lipids and lipid globules, gels, immiscibleparticles, blastomeres, aggregations of cells, microorganisms, and otherbiological materials. For example, the object selection in accordancewith the present disclosure may include cell “washing”, in whichcontaminants (such as bacteria) are removed from cellular suspensions,which may be particularly useful in medical and food industryapplications. Further, the present invention has the applicability toselect non-motile cellular objects from motile cellular objects.

The subject matter of the present disclosure may also be utilized totransfer a species from one solution to another solution whereseparation by filtering or centrifugation is not practical or desirable.In addition to the applications discussed above, additional applicationsinclude selecting colloids of a given size from colloids of other sizes(for research or commercial applications), and washing particles such ascells, egg cells, etc. (effectively replacing the medium in which theyare contained and removing contaminants), or washing particles such asnanotubes from a solution of salts and surfactants with a different saltconcentration or without surfactants, for example.

The action of selecting species may rely on a number of physicalproperties of the objects or objects including self-motility,self-diffusivity, free-fall velocity, or action under an external force,such as an actuator, an electromagnetic field, or a holographic opticaltrap. The properties which may be selected include, for example, cellmotility, cell viability, object size, object mass, object density, thetendency of objects to attract or repel one another or other objects inthe flow, object charge, object surface chemistry, and the tendency ofcertain other objects (i.e., molecules) to adhere to the object.

While discussion below focuses on the identification and selection ofviable or motile sperm from non-viable or non-motile sperm, or selectingsperm by gender and other sex selection variations, or selecting one ormore labeled cells from un-labeled cells distinguishingdesirable/undesirable traits, etc., the apparatus, methods, and systemsof the present invention may be extended to other types of particulate,biological or cellular matter, which are capable of being interrogatedby fluorescence techniques within a fluid flow, or which are capable ofbeing manipulated between different fluid flows into one or moreoutputs.

Sample Preparation: In one embodiment, a concentration of objects 160,such as cells (i.e., raw semen), is determined using a cell countingdevice, such as a Nucleocounter. In one embodiment, the appropriatestaining volume is obtained (i.e., using staining a calculatorworksheet), and the volume of staining TALP and objects 160 (i.e., neatsemen) that need to be added for a predetermined cell concentration(i.e., 200×106/ml sperm concentration), are calculated. For example, a 1ml amount of stained sample is prepared when the neat semenconcentration=1500×106/ml. Thus, 200×106/ml/1500×106/ml=0.133 ml neatsemen, which is added to (in order) 0.012 ml Hoechst 33342 (5 mg/mlstock solution), and 0.855 staining TALP (pH 7.4), to equal 1 ml totalstaining volume at 200×106/ml.

In one embodiment, staining TALP is prepared by filling a container(i.e., beaker) with Milli-Q water to ⅔ of the total desired volume. Astir bar and stir plate are used to mix the solution as chemicals areadded. The chemicals, which are added in the order listed (up to theGentamicin, which is added later), include:

TABLE 1 Chemical components of staining TALP CHEMICAL FORMULA g/100 mlg/500 ml g/1000 ml HEPES C₆H₁₂N₂O₄S 0.952 4.760 9.520 MagnesiumMgCl₂*6H₂O 0.008 0.040 0.080 Chloride 6- Hydrate, crystal Sodium NaCl0.5518 2.759 5.518 Chloride Potassium KCl 0.0224 0.116 0.224 ChlorideSodium Na₂HPO₄ 0.004 0.020 0.040 Phosphate Dibasic/ Anhydrous SodiumNaHCO₃ 0.084 0.420 0.840 Bicarbonate Pyruvic Acid Na Pyruvate 0.0220.110 0.220 Glucose C₆H₁₂O₆ 0.090 0.450 0.900 Lactic Acid, Na Lactate0.361 1.805 3.610 60% Syrup (ml) Bovine Serum BSA 0.300 1.500 3.000Albumin Gentamicin 0.25 ml 1.25 ml 2.50 ml Solution (10 mg/ml) AFTERFILTRATION

In one embodiment, after adequate mixing of the chemicals, the pH isadjusted to 7.4 using NaOH. Additional Milli-Q water is used to bringthe solution to a final volume in a container (i.e., volumetric flask).A sterile filter (i.e., 0.22 sterile filter) is used to filter/sterilizethe volume. An antibiotic (i.e., Gentamicin solution) is added afterfiltration, and the volume of staining TALP is stored at 5° C. and canbe used for 7-10 days.

Thus, after the volume of sample is stained with staining TALP, in oneembodiment, the stained samples 120 are placed in containers (i.e.,tubes) into a water bath set at 34-35 CC, and incubated for apredetermined time (i.e., 45 minutes). In one embodiment, afterincubation, the stained samples are removed from the water bath, and anequal volume of 4.0% egg-yolk TALP with red food dye that has beenwarmed in the water bath set to 34-35° C., is added.

To obtain 4% egg yolk TALP with red food dye, the staining TALP as notedabove is prepared, and a desired volume of final solution is determined.The volume of staining TALP and egg yolk required to prepare a 4% eggyolk solution is calculated as follows:

The desired volume: (250 ml)×0.04=10 ml egg yolk needed for 4% solution.240 ml of staining TALP is added to a graduated container (i.e.,cylinder), and 10 ml of egg yolk is added. FD&C #40 food dye is added tothe container (i.e., cylinder), to obtain: 0.261 ml/100 ml of solution.With a desired 250 ml total volume, 0.261 ml×250 ml/100=0.653 ml of redfood dye. The container (i.e., cylinder) is covered with parafilm andcarefully inverted until the volume is thoroughly mixed. The volumecontainer is then allowed to sit overnight and cooled in a cool room.The volume container is then carefully decanted into a sterilecontainer, leaving any sediment at the bottom of the volume container.The volume is then filtered/sterilized through a 0.22 .tm bottle topfilter, and an appropriate amount of antibiotic solution is added (i.e.,0.250 ml Gentamicin/100 ml of egg yolk TALP).

Thus, after the addition of an equal volume of 4% egg-yolk TALP with redfood dye to the stained sample 120, the stained samples 120 are filteredby pouring the samples 120 into a 20-micron filter (i.e., CellTricsfilter), that drains into another sterile 5 ml culture container. Aftera predetermined time of staining (i.e., 45 minutes), an equal volume of4% egg yolk TALP is added. The stained sample 120 (i.e., cells) are runthrough a filter (i.e., Partec filter, with 50-micron mesh), and thesample is placed into a sample holder or reservoir 233 for introductioninto the microfluidic chip 100 (see FIGS. 19-21 ).

In one embodiment, the final sperm concentration=100×106/ml, and thefinal egg-yolk percentage=2%. A new sample aliquot 120 can be preparedand used every hour if desired.

Microfluidic Chip System: The various embodiments of the microfluidicchip, as described below, utilize one or more flow channels, having aplurality of substantially laminar flows, allowing one or more objectsto be interrogated for identification by an interrogation apparatus, andto be acted upon by a focused energy apparatus, with the objects exitingthe microfluidic chip into one or more outputs. In one embodiment, theobjects not targeted by the focused energy apparatus are undisturbed,and the focused energy apparatus photodamages, alters, disables, kills,or destroys targeted objects.

The various embodiments of the present invention thereby provide aselection of objects on a continuous basis, such as, within acontinuous, closed system without the potential damage and contaminationof prior art methods, particularly as provided in sperm separation. Thecontinuous process of the present invention also provides significanttime savings in selecting and discriminating objects.

While the present subject matter is discussed in detail with respect toa microfluidic chip 100 illustrated in FIGS. 1A-2 and a microfluidicchip holder 200 illustrated in FIGS. 20-21 , it should be understoodthat this discussion applies equally to the various other embodimentsdiscussed herein or any variation thereof.

Microfluidic Chip: FIG. 1A is an illustrative embodiment of amicrofluidic chip 100. The microfluidic chip 100 is manufactured of asuitable material such as glass, or a thermoplastic (e.g., lowauto-fluorescing polymer, etc.), or combination of materials, through anembossing process, soft photolithography, or injection molding process,as well known to one of ordinary skill in the art, and is of suitablesize. Each layer may be any suitable thickness, for example, thethickness may be within a range of approximately 300-400 μm, and morepreferably the thickness may be approximately 400 μm.

The microfluidic chip 100 includes one or more structural layers inwhich are disposed micro-channels which serve as sample inputchannel(s), sheath or buffer fluid channel(s), output channel(s), etc.The micro-channels are of suitable size to accommodate laminar flowstreams containing objects, and may be disposed in any of the layers ofthe chip 100 in the appropriate length, as long as the object of thepresent invention is realized. In one embodiment, the dimensions of themicrofluidic channels range from 50 microns to 500 microns, with 100-300microns being preferably used to avoid clogging.

The desired flow rate through the microfluidic chip 100 may becontrolled by a predetermined introduction flow rate into the chip 100,maintaining the appropriate micro-channel dimensions within the chip100, by pumping mechanisms that pump external fluids into the chip 100,and by providing narrowing or tapering of the micro-channels at variouslocations, and/or by providing obstacles, ramps, or dividers within themicro-channels (further discussed below).

More specifically, a plurality of inputs is provided into themicrofluidic chip 100, which inputs provide access to themicro-channels/channels. In one embodiment, as shown in FIGS. 1A-2 , asample input 106 is used for introducing a sample of particles orobjects (i.e., cells) 160 in a sample fluid mixture 120 into a mainfluid channel 164 of the microfluidic chip 100 from at least onereservoir source (see FIG. 19 ).

The microfluidic chip 100 also includes at least one sheath or bufferinput for the introduction of sheath or buffer fluids. In oneembodiment, there are two sheath or buffer inputs in the microfluidicchip 100, which include a sheath or buffer input 107 and sheath orbuffer input 108, both disposed proximate to the sample input 106, andwhich both introduce sheath or buffer fluids 163 into the microfluidicchip 100 (see FIGS. 1A-3B).

In one embodiment, there are three sheath or buffer inputs 107, 108, and172 (see FIGS. 1A and 3B) which introduce sheath or buffer fluids intothe channel 164 of the microfluidic chip 100. The location of the sheathor buffer inputs 107, 108, 172 may vary, and they may access channels inthe chip 100 which are in the same or different structural layers. Inone embodiment, the sheath or buffer fluids 163 are introduced intoinputs 107, 108, 172 from a common reservoir (see FIGS. 20-21 ), or inanother embodiment, from separate reservoirs (see FIG. 19 ).

The sheath or buffer fluids are well known in the art of microfluidics,and in one embodiment, may contain nutrients well known in the art tomaintain the viability of the objects 160 (i.e., sperm cells) in thefluid mixture. Commercially available Tris, as sold by Chata Biosystems,is one example, and the sheath or buffer fluid 163 may be formulated toinclude the following: Water—0.9712 L; Tris—23.88 gg; citric acidmonohydrate—11.63 g: D-fructose—8.55 g. The pH is adjusted to 6.80±0.05with hydrochloric acid, and osmolarity is adjusted, if necessary, to270-276 mOsm with fructose high purity. The mixture is filtered using a0.22-micron filter.

The microfluidic chip 100 may have one or more structural layers inwhich the micro-channels are disposed. The channels may be disposed inone or more layers or in-between layers. The following embodimentsdescribe a bonding process, but one of ordinary skill in the art wouldknow how to achieve the various features by using an injection moldingprocess. For example, in injection molding, instead of forming twolayers, two molds could be made and joined together, such that aninjection is made into the cavity in order to obtain the chip of thepresent invention.

In one embodiment, as shown in FIG. 1A, one structural layer 101 isincluded in the microfluidic chip, with a top, “blank” plastic layer 104disposed thereon. The top, “blank” layer 104 bonds with the functionallayer 101 to form an enclosed microfluidic network, and may havemultiple holes to provide access to the lower layer(s) of the chip 100.For example, the top “blank” layer 104 may have holes corresponding toinputs 106, 107, 108, 172, etc., or provide holes 145 for pins to securethe layers 101, 102, 104, etc., of the chip 100 together. In oneembodiment, the top layer 104 of the microfluidic chip 100 includes aplurality of apertures configured to align with the fittings on amicrofluidic chip holder 200 (further described below).

In another embodiment, as shown in FIG. 1 B (a), two functional,structural plastic layers 101-102 are included in the microfluidic chip100, with no top, “blank” layer. In this embodiment, the functional sideof the top layer 102 is disposed on the underside of the layer 102, sothat when the layers are put together, the channels 114, 115, 116, 117are formed (see FIG. 1 B (b).

In yet another embodiment, as shown in FIG. 1C (a), three functional,structural layers 101, 102, 103 are used in the microfluidic chip 100.As with FIG. 1B (a), layers 102, 103 include functional sides on theundersides of the layers 102, 103, with layers 101 and 102 formingchannels 116, 117 when put together. Layer 103 has channels 114, 115disposed on the underside of the layer 103 (see FIG. 1C (b)).

In yet another embodiment, as shown in FIG. 1D, four structural plasticlayers 101-103 and a top, “blank” layer 104, are used in themicrofluidic chip 100. In this embodiment, layer 102 includes 114, 115,and layers 101 and 103 each include one of channels 117, 116,respectively.

However, one of ordinary skill in the art would know that more or fewerstructural layers with functional sides, and with or without “blank”layers, may be used, and the channels may be disposed in any of thestructural layers, or in different structural layers, and in anyarrangement, with access to those channels through a top “blank” layer,as long as the object of the present invention is achieved.

In one embodiment, a sample fluid mixture 120 including objects 160, isintroduced into sample input 106, and the fluid mixture 120 flowsthrough main channel 164 toward action chamber 129 (see FIGS. 1A-2 ).The sheath or buffer fluids 163 are introduced into sheath or bufferinputs 107, 108 (see FIGS. 1A-2 ) in most embodiments, and into sheathor buffer inputs 107, 108, and 172 in another embodiment (see FIG. 1A).The sheath or buffer fluids 163 flow-through channels 114, 115 and 116,117, into the main channel 164, and towards the action chamber 129before flowing out through at least output channels 140 and 142, inlaminar flow.

In one embodiment, the fluid mixture 120 from main channel 164 joinswith the sheath or buffer fluids 163 from channels 114, 115 atintersection 161 of the microfluidic chip 100. In one embodiment, bufferfluids 163 from channels 116, 117 join the combined fluid mixture 120and sheath or buffer fluids 163 from first intersection 161, downstreamat second intersection 162 (see FIGS. 1A-2 ). In one embodiment, sheathor buffer fluids 163 are inputted via input 172 into main fluid channel164, downstream from the second intersection 162 (see FIGS. 1A and 3B).

In one embodiment, channels 114, 115 are substantially the samedimensions as channels 116, 117, as long as the desired flow rate(s) isachieved to accomplish the object of the present invention, but one ofordinary skill in the art would know that the dimensions may bedifferent as long as they accomplish the desired results (furtherdiscussion below).

In one embodiment, channels 114-117 and 140-142 may have substantiallythe same dimensions, however, one of ordinary skill in the art wouldknow that the size of any or all of the channels in the microfluidicchip 100 may vary in dimension (for example, between 50 and 500microns), as long as the desired flow rate(s) is achieved to accomplishthe object of the present invention. In one exemplary embodiment, thechannels 114, 115 or 116, 117 are disposed in the same structural layeror plane of the microfluidic chip 100 as the layer or plane in which thechannel 164 is disposed (see FIG. 1A, for example), or may be disposedin a different structural layer or plane (see FIG. 1B, for example). Inanother embodiment, the input channel 164 and the sheath channels 114,115 or 116, 117, may be disposed in-between structural layers or planesof the chip 100. Thus, one of ordinary skill in the art would know thatthe channels 114-117, 164, and 140-142, etc., can be disposed in anylayer or between any two layers. Further, although the channels 114-117,164, and 140-142, etc. are described in exemplary embodiments as shownin the Figures, one of ordinary skill in the art would know that theparticular arrangement or layout of the channels on the chip 100 may bein any desired arrangement as long as they achieve the describedfeatures of the present invention.

In one embodiment, the channels 116, 117 are cut through layer 101 (seeFIGS. 1A and 2 ), and join the fluid mixture 120 in channel 164 in thesame plane, via holes cut through the layers. In one embodiment,channels 116, 117 substantially parallel input channel 164, and eachjoin intersection 161 at an angle from channel 164 (see FIGS. 2-3 ). Thesheath or buffer fluids from channels 116, 117 compress the fluidmixture 120 flow horizontally from the sides, or laterally, such thatthe objects 160 in the fluid mixture 120 are flattened and/or orientedin a selected or desired direction, while still maintaining laminar flowin channel 164 (i.e., the first in two steps of hydrodynamic focusing,as described further below).

Further, in one embodiment, channels 114, 115 join the fluid mixture 120in channel 164 at intersection 162, with each channel 114, 115 at anangle from channel 164 (see FIG. 1A). The sheath or buffer fluids fromchannels 114, 115 compress the fluid mixture 120 flow with respect tochannel 164 (see FIG. 14 ), such that the objects 160 in the fluidmixture 120 are further flattened and/or oriented in the selected ordesired direction, while still maintaining laminar flow in channel 164(i.e., the second in two steps of hydrodynamic focusing, as describedfurther below).

Further to this embodiment, a third sheath or buffer fluid input 172 isdisposed downstream from intersection 162 (see FIG. 3B), which allows athird hydrodynamic focusing step to take place, where the sample fluidmixture 120 is compressed from above the channel 164 by the sheath orbuffer fluids 163 introduced therein.

In an alternative embodiment, after the first hydrodynamic focusing stepdescribed above, the channels 114, 115 join the fluid mixture 120 inchannel 164 at intersection 162 from an angle from above (see FIG.3A)—and may be above and below (see FIG. 12A)—channel 164, to compressthe fluid mixture 120 from a vertical direction to further flattenand/or orient the objects 160 in the channel 164 (i.e., the secondhydrodynamic focusing step).

However, one of ordinary skill in the art would appreciate that thedepicted configurations, angles, and structural arrangements of themicrofluidic chip 100 sheath or buffer inputs, sample input, and sampleinput channel and sheath or buffer channels, as well as the hydrodynamicfocusing steps, may be different as long as they achieve the desiredfeatures of the present invention.

In one embodiment, as shown in FIG. 2 , channels 114, 115, and 116, 117are depicted as partially coaxial to one another with a center pointdefined by the sample input 106. Thus, in one embodiment, channels 114,115, and 116, 117 are disposed in a substantially parallel arrangement,with the channels 114, 115, and 116, 117 being equidistant to mainchannel 164. However, one of ordinary skill in the art would recognizethat the depicted configuration may be different as long as it achievesthe desired features of the present invention.

In one embodiment, holes and pins/posts 145 are disposed at variousconvenient positions in the layers 101, 102, 103, 104, etc., to fix andalign the multiple layers during chip 100 fabrication.

In one embodiment, a gasket 105 of any desired shape, or O-rings, may beprovided to maintain a tight seal between the microfluidic chip 100 andthe microfluidic chip holder 200 (see FIGS. 1D and 21-22 , for example).In the case of a gasket 105, it may be a single sheet or a plurality ofobjects, in any configuration, or material (i.e., rubber, silicone,etc.) as desired. In one embodiment, as shown in FIG. 1D, a first gasket105 is disposed at one end of the microfluidic chip 100 and interfacesor is bonded with layer 104. A plurality of holes 144 are provided inthe first gasket 105 and are configured to align with the sample input106, sheath/buffer input 107, and sheath/buffer input 108.

In one embodiment, a second gasket 143 may be disposed at another end ofthe microfluidic chip 100 opposite to the first gasket 105 (see FIG. 1D,for example), and interfaces or is bonded with (using epoxy) the topstructural layer 104 (see FIGS. 1D and 21-22 ).

In one embodiment, O-rings are used instead of gaskets, to assist insealing, as well as stabilizing the microfluidic chip 100 in the chipholder 200.

However, one of ordinary skill in the art would know that one or moregaskets or O-rings may be applied to the outer layers of the chip 100 inorder to protect the chip 100 in a chip holder 200, during operationthereof.

In one embodiment, the channels 114-117, and 140-142, of themicrofluidic chip 100, may not just vary in dimension but may havetapered shapes at entry points to other channels in the chip 100 inorder to control the flow of fluid through the channels. For example,main channel 164 may taper at the entry point into intersection 161 (seeFIG. 4A, taper 166A), or at the entry point into intersection 162 (seeFIG. 4B, taper 166A) to control and speed up the flow of sample 120 intothe intersection 161, and allow the sheath or buffer fluids 163 fromchannels 116, 117 or 114, 115, respectively, to compress the samplefluid mixture 120 in a first direction (i.e., horizontally or laterally)on at least two sides, if not all sides (depending on where the fluidchannel 164 enters the intersection 161), and in a second direction(i.e., vertically) (see FIGS. 3A-3B, 4A-43 , and tapers 166A).

In another embodiment, ramps may be disposed in channel 164 or channels114-117 to achieve the effect of controlling and speeding up the sampleflow through the channels. The ramps may be in addition or instead oftapers.

For example, a ramp 1663 may be disposed in channel 164 prior to thesample flow approaching intersections 161 and 162, respectively, orprior to entering action chamber 129 (see FIGS. 4A and 4B).

Thus, the sample fluid mixture 120 becomes a relatively smaller,narrower stream, bounded or surrounded by sheath or buffer fluids 163,while maintaining laminar flow in channel 164. However, one of ordinaryskill in the art would know that the main channel 164, or the bufferchannels 114-117 may be of any physical arrangement, such as arectangular or circular-shaped channel, with tapers, ramps, or otherinternal features, as long as the object of the present invention isobtained.

In one embodiment, a plurality of output channels stemming from mainchannel 164 (see FIG. 2 ) is provided for removal of fluid flowedthrough the microfluidic chip 100, including any targeted ornon-targeted objects 160 and/or sheath or buffer fluids 163. In oneembodiment as shown in FIGS. 1A-2 , there are three output channels140-142 which include a left side output channel 140, a center outputchannel 141, and a right side output channel 142. The left side outputchannel 140 ends at a first output 111, the center output channel 141ends at a second output 112, and the right side output channel 142 endsat a third output 113. However, it is possible to have only one outputchannel 141, and output 112.

In one embodiment, output channels 140-142 depart from channel 164within chamber 129 to outputs 111-113. In one embodiment, thecross-section and the length of the output channels 140-142 should bemaintained at a predetermined volume ratio (i.e., 2:1:2, or 1:2:1, etc.)to obtain the desired hydraulic resistance of the output channels140-142.

In one embodiment, the output channels 140-142 increase in dimensionfrom the channel 164, leaving the chamber 129, such that the outputratio for the objects 160, is increased through the relevant channel141.

In one embodiment, instead of a straight edge, where necessary, aplurality of notches or recesses 146 may be disposed at a bottom edge ofthe microfluidic chip 100 to separate the outputs (i.e., outputs111-113) and for the attachment of containers, and external tubing (forrecycling the sheath or buffer fluids 163—see FIGS. 19-21 ), etc. Thefirst output 111, the second output 112, and the third output 113 arereached via output channels 140-142 which originate from action chamber129 (see FIG. 2 ).

In one embodiment, a container 188 collects the objects 160 from thesecond output 112, although other containers may collect the output fromfirst output 111 and third output 113 (see FIGS. 6A-6D). In oneembodiment, portions of the first, second, and third outputs 111-113 maybe characterized electronically, to detect concentrations of objects160, pH measuring, cell 160 counts, electrolyte concentration, etc.

In one embodiment, the targeted objects 160 are acted upon by thefocused energy apparatus 157, and those objects 160, as well asnon-targeted objects 160, may be collected as product 165 from thesecond output 112.

In one embodiment, the product 165 of targeted and non-targeted objects160 may continue to be processed for storage, for further separation, orfor processing, such as cryopreservation (discussed further below).

In one embodiment, the microfluidic chip 100 is provided in a sterilestate, and may be primed with one or more solutions (i.e., sheath orbuffer fluids 163), or purged of any fluids or materials by eitherdraining the microfluidic chip 100 or by flowing sheath or buffer fluids153 or other solutions through the microfluidic chip 100, according toknown methods.

Action Chamber: In one embodiment, downstream from intersection 162, theobjects 160 in the fluid mixture 120 flow-through channel 164 into anaction chamber 129, where the objects 160 are interrogated and actedupon. In one embodiment, channel 164 tapers into the chamber 129 (seeFIG. 4B), which speeds up the flow of the fluid mixture through thechamber 129. However, one of ordinary skill in the art would know thatthe channel 164 need not taper and could be of any dimension and size aslong as the present invention performs according to the desiredrequirements.

In one embodiment, an interrogation apparatus 147 is used to interrogateand identify the objects 160 in the fluid mixture in channel 164 passingthrough the chamber 129. Further, in one embodiment, the focused energydevice 157 also acts upon the objects 160 passing through the chamber129.

In one embodiment, the chamber 129 includes a relatively small diameteropening or window 150 (see FIG. 5 ) cut through the microfluidic chip100 and layers 101-102, through which the objects 160 can be visualizedas they pass through channel 164.

Further, a shallow opening with a relatively larger diameter is cut intolayer 104 as a top window, and into layer 101 as a bottom window. In oneembodiment, the top window is configured to receive a first transparentcovering 133, and the bottom window 152 is configured to receive asecond transparent covering 132. The coverings 133, 132 may be made ofany material with the desired transmission requirements, such asplastic, glass, or may even be a lens. In another embodiment, instead ofa window with coverings, a contiguous plastic sheet can be used. Notethat although the relative diameters of the coverings 132, 133, andopening 150 are shown in FIG. 5 , these may vary according to design ormanufacturing considerations.

In one embodiment, the above-mentioned first and second coverings 133,132 are configured to enclose the chamber 129. The windows and coverings133, 132 (see FIG. 5 ), allow the objects 160 flowing in the fluidmixture 120 in channel 164 through the chamber 129, to be viewed throughopening 150, and acted upon by a suitable light source 147 and a focusedenergy apparatus 167 (discussed later).

In one embodiment, windows and/or openings are not required due to thestructure of the chip layers (i.e., glass), and/or their configuration,or the focused energy device 157 and light source 147 wavelengths andpower levels are such that no damage to the chip 100 will occur.

Interrogation Apparatus: The interrogation apparatus of the presentinvention includes a light source 147 which is configured to emit ahigh-intensity beam 148 with any wavelength that matches excitableobjects in the fluid mixture 120 (see FIG. 5 ). Although a laser 147 ispreferred, any suitable other light sources 147 may be used, such as alight-emitting diode (LED), or arc lamp, etc. to emit a beam thatexcites the objects.

In one embodiment, such a high-intensity laser beam 148 from a suitablelaser 147 of a preselected wavelength—for example, a 349 nm or 355 nmcontinuous wave (CW), or quasi-CW pulsed laser 147—is required to excitethe objects 160 in the fluid mixture (i.e., sperm cells). In anotherembodiment, a 532 nm green laser 147 is utilized.

In one embodiment, the laser 147 (see FIG. 5 ) emits a laser beam 148through the covering 133 at an uppermost portion of the chip 100,through opening 150, to illuminate the objects 160 flowing throughchannel 164 in chamber 129 of the chip 100, and then through covering132 in layer 101 of the chip 100.

In one embodiment, the light beam 148 can be delivered to the objects160 by an optical fiber that is embedded in the microfluidic chip 100 atopening 150.

The high-intensity beam 148 interacts with the objects 160 (see detailedexplanation below), and passes through the first coverings 133, to exitfrom the covering 132 at the bottom window, such that the emitted light151, which is induced by the beam 148, is received by an objective lens153 or other collection optics. The objective lens 153 or othercollection optics may be disposed in any suitable position with respectto the microfluidic chip 100—for example, parallel to the main channelwith the optical axis perpendicular to the sample fluid flow 120.Because the chamber 129 is sealed by the first and second coverings 133,132, the high-intensity beam 148 does not impinge on the microfluidicchip 100 and damage the layers 101,104 (see FIG. 1A). Thus, the firstand second coverings 133, 132 help prevent damage to the microfluidicchip 100 from the high-intensity beam 148 and photonic noise inducedfrom the microfluidic chip 100 material (i.e., plastic).

In one embodiment, the light beam 148 passes through the chip 100 andthe emitted light 151 received by the objective lens 153 or othercollection optics, is detected by detector 154, and is converted into anelectronic signal by an optical sensor 154, such as a photomultipliertube (PMT) or photodiode, etc. The electronic signal can be digitized orprocessed by an analog-to-digital converter (ADC) 155 and sent to adigital signal processor (DSP) based controller 156 or computer. Theelectronic controller 156 can be any electronic processor with adequateprocessing power, such as a DSP, a Micro Controller Unit (MCU), a FieldProgrammable Gate Array (FPGA), or even a Central Processing Unit (CPU).

In one embodiment, the DSP-based controller 156 monitors the electronicsignal and based upon predetermined criteria, the focused energyapparatus 157 may be employed when a targeted object 160 is detected.

However, in another embodiment, the interrogation apparatus simplyinterrogates the objects 160 in the sample fluid flow 120 for identity,and it is not connected to the employment of the focused energyapparatus 157 (see FIG. 6C).

Focused Energy Apparatus: In one embodiment, in order to deliver adesired energy level to objects 160, a focused energy apparatus 157 isused to provide focused energy pulses to the objects 160. The focusedenergy apparatus 157 may be a thermal, electrical, optical, orelectromagnetic device 157, which would have a desired wavelength, andwould deliver high peak power with a very high repetition rate (or pulsefrequency), to the target objects 160.

In one embodiment, the focused energy apparatus 157 is triggered apredetermined time (i.e., milliseconds) after activation by thecontroller 156 (the timing being set based upon the traveling speed ofobjects 160 through the channel 164, and is discussed further below),and issues a pulse to the selected or targeted (i.e., unwanted) object160.

Examples of pulsed lasers 157 include mode-locked, Q-switch, as well asthose lasers using both mode-locking and Q-switch techniques. Forexample, a focused energy device 157 such as an Avia 355-5-100 (made byCoherent, Inc., Santa Clara, Calif.), or the Explorer XP lasers Q-switchlaser from Spectra-Physics Inc., is capable of operating in apulse-on-demand mode, and can deliver 15 ns energy pulses or less, at arate of over 1000 pulses per second, to the target objects 160.

In one embodiment, pulse energy levels of 0.5-8.0 pJ are used, and in apreferable embodiment, a Q-switch laser 157 in pulse-on-demand mode isused to deliver an average pulse energy of 1.8 pJ with a range forindividual pulses of 1.3 pJ to 2.3 μJ. In one embodiment, the pulsewidth ranges from 3 nanoseconds to 1 microsecond, and preferably, is ina range of 5-9 nanoseconds. However, one of ordinary skill in the artwould know that any high power laser existing now, or later developed,with the appropriate high energy pulses and pulse frequency, would besuitable for the present invention in order to achieve the desiredtarget accuracy and/or effect.

In one embodiment, the need for a tight action region (i.e., chamber129, or space between the chip 100 and container 188) in order todeliver the pulsed energy from the focused energy apparatus 157 totarget objects 160 or a surrounding region thereof, is important tominimize the potential impact of delivering the energy outside of thetargeted objects 160 or region, to otherwise unselected, or non-targetedobjects 160. For example, a focused energy apparatus 157 such as theExplorer XP 355-1 Q-switch laser, is capable of delivering <4% rms,providing high pulse-to-pulse stability when fired at regular uniformintervals.

However, for flow cytometric analysis and action systems where objects160 or cells enter the action region (i.e. chamber 129, or space betweenthe chip 100 and container 188—see FIGS. 6A-D) in non-uniform intervals,additional measures are employed to deliver uniform pulse energy 158 toimpinge only the targeted objects 160 or cells, or the surroundingregion thereof. Such measures include matching laser 157 performanceparameters such as pulse length and peak power levels, to enable thesystem of the present invention to achieve a desired target accuracy(i.e., in one embodiment, photodamage, or kill rate of 95% or higher hitrate on target objects 160).

In addition, further tuning the pulse-on-demand operation andperformance of the laser 157 to deliver extremely high pulse-to-pulsestability when fired at non-uniform intervals, greatly reduces thespatial variability in the area impacted by the pulse 158. Thus, byreducing pulse-to-pulse variability in the focused energy device 157,the unintended action, damage, or destruction to non-target objects 160or cells, is greatly reduced, achieving, for example, an 85% or higherrate of viability for live, non-target objects 160 or cells.

In one embodiment, the focused energy apparatus 157 is utilized in anaction region 129, such as chamber 129, prior to interrogation by theinterrogation apparatus 157 (see FIG. 6D), and in another embodiment,the focused energy apparatus 157 is utilized in the action region (i.e.,chamber 129) after interrogation by the interrogation apparatus 157 (seeFIG. 6A). In yet another embodiment, the focused energy apparatus 157acts upon the sample fluid with objects 160 after it leaves the chip 100and enters a container 188—either at the output 112 or in disconnecteddroplet form 187 before it enters the container 188 (see FIGS. 6B-C).

In the embodiment where the focused energy apparatus 157 acts upon theobjects 160 after they are interrogated by the interrogation apparatus147 in the action region 129 (i.e., chamber 129), upon determinationthat the objects 160 are to be targeted, the focused energy apparatus157 emits a focused energy beam 158 to act upon the objects 160 flowingthrough channel 164 (see FIGS. 5 and 6A-6C, for example).

In the embodiment, the focused energy apparatus 157 acts upon theobjects 160 after interrogation in action region 129, and after theobjects 160 flow through output channel 141, but before the sample fluid120 is collected by container 188. In this embodiment, the focusedenergy apparatus 157 is utilized as above but is positioned to emit thebeam between the chip 100 and the container 188. In one embodiment thesample fluid 120 simply falls from output 112 through the air into thecontainer 188 in droplet form 187, and in another embodiment, there maybe a transparent enclosure between the chip 100 and the container 188.

The focused energy apparatus 157 can be set to damage, alter, disable,kill or destroy the targeted or unwanted object 160 in the sample fluid120 with a pulse, or to activate one of several mechanisms in the object160 or cell, such that cell damage or death ensues.

However, depending on the desired arrangement (see further below forvarious embodiments), the targeted or selected objects 160 may be wantedobjects 160, in which case the focused energy apparatus 157 is notactivated or triggered, or the targeted or selected objects 160 may beunwanted objects 160, where the focused energy apparatus 167 isactivated act upon the objects 160, such as to damage, alter, disable,kill or destroy the selected, unwanted objects 160. However, these arenot the only embodiments, and the various embodiments are discussedfurther below.

In one embodiment, when the selected object 160 is damaged, altered,disabled, killed, or destroyed by the focused energy device 157, theobject 160 continues to flow through the main channel 164 to the centeroutput channel 141, and to the second output 112, and into container188, along with any non-targeted objects 160. The sheath or bufferfluids 163 proceed in laminar flow through output channels 140, 142, tooutputs 111, 112, respectively.

However, in one embodiment, as noted above, the objects 160 in channel164 may flow out from the chip 100 through output channel 141 and singleoutput 112.

Accordingly, in one embodiment, the present methods and apparatus arecapable of producing a discriminated product 165 (see FIGS. 6A-6D) ofobjects 160 in container 188, including a high viability of non-targetor wanted objects 160 or cells, and a high percentage of photodamaged,altered, disabled, destroyed, or dead target objects 160.

Beam Shaping and Optics: In order to achieve satisfactory signalrepetition and efficient damaging, altering, disabling, killing, ordestruction of objects 160, it is advantageous to use beam shapingoptics for both the interrogation beam 148 and the focused energy beam158 (see FIGS. 16-17 ). As used herein, the phrase “beam spot” refers toa cross-section of either beam 148, or beam 158.

In one embodiment, the focused energy apparatus 157 is disposeddownstream from a light source 147, and on the same side as the lightsource 147 (see FIG. 17 ), but the focused energy apparatus 157 may alsobe disposed downstream and on an opposite side of the light source 147(see FIG. 16 , for example).

In the embodiment of FIG. 16 , a beam shaping optics for theinterrogation beam 148 is disposed on one side of chip 100. Theinterrogation beam 148 from light source 147 passes through the actionregion (i.e., chamber 129) and is received by objective lens 153.

In one embodiment, the beam 148 is expanded by the beam shaping optics181, which may include a plurality of lenses, which arrangement would bewell known to one of ordinary skill in the art. For example, the beamshaping optics 181 may include a pair of prisms and a pair ofcylindrical lenses with appropriate focal lengths or may include otherlenses, with or without prisms, which would be available to one ofordinary skill in the art. The beam expansion enables the final spotsize at the focal point in the interrogation region 129. In oneembodiment, the circular beam 148 spot is expanded using a beam expander180. The beam expansion also reduces the influence on the downstreamoptics, limiting damage and extending lifetime. However, in oneembodiment, no beam expander is utilized. Alternatively, if the sourcebeam has too large of a diameter, optics could be used to reduce thatdiameter to a suitable size.

In one embodiment, the beam shaping optics 181 include two perpendicularcylindrical lenses to alter the beam shape 148 into an ellipseperpendicular to the direction of sample fluid 120 flow, and along thedirection of sample fluid 120 flow, when focused at the center thereof.This elliptical beam 148 spot serves to excite the objects 160 passingthrough the channel 164 of the microfluidic chip 100, and providesmaximum uniform illumination at a center area of the beam 148 spot, tocompensate for minor fluctuations in the flow of objects 160 through thechannel 164. Further, in one embodiment, the ellipse of the beam shapehaving a wider dimension perpendicular to the sample fluid 120 flow,helps to reduce variation in the fluorescence signal coming from theobjects 160 (i.e., sperm cells) that are not perfectly centered withinthe sample fluid 120 flow stream. The narrow dimension keeps the beam148 at a high enough intensity to adequately excite the fluorescent dyefor interrogation of the objects 160 (i.e., sperm cells). While anelliptical beam 148 spot is preferred, in other embodiments of thepresent invention, a different shaped beam may be utilized. The power ofthe interrogation beam can be adjusted as well to assist in theinterrogation and to limit the impact on the interrogated objects

In one embodiment, the focused energy beam 158 is also shaped by beamshaping optics 180. The shape of the focused beam 158 spot of thefocused energy device 157 influences the desired target accuracy andpotential for impacting non-target objects 160 in the channel 164, andcan be varying beam shapes, as the application requires. In a flow-basedsystem, the beam width along the direction of flow of the sample fluid120 should be adjusted to be sufficiently narrow such that only thetarget object 160 is affected, and sufficient beam intensityconcentration is achieved. The length of the beam 158 spot across thefluid channel 164 can be intentionally adjusted to compensate for anyslight instability and variability in the focused flow of the samplefluid 120. Desired beam shaping can easily be achieved by one skilled inthe art.

In one embodiment, as shown in FIG. 16 , beam shaping optics 180 for thefocused energy beam 158 is utilized to focus the beam 158 down to a muchsmaller size to increase a laser flux in the action region 129. In oneembodiment, the beam 158 is expanded by beam expander optics 180 (seeFIG. 16 ) which may include a plurality of lenses or prisms withappropriate focal lengths, which would be readily known to one ofordinary skill in the art.

In one embodiment, the beam 158 passes through a pair of anamorphicprisms, for example, to shape the beam 158. The beam 158 is furtherfocused and compressed in the horizontal and vertical directions by anoptical object due to a Gaussian beam property. In one embodiment, theoptical object may be, for example, detector optics 153 such as amicroscope objective or a focusing lens with a short focal length. Thebeam spot provides a combination of energy concentration for efficientaction (i.e., killing, etc.) and sufficient width to compensate forminor fluctuations in a flow of the objects 160 through the microfluidicchannel 164. In one embodiment, a pair of cylindrical lenses is used toexpand the beam in the vertical dimension, before a spherical focusinglens or objective lens is used to focus the beam to an elliptical beamspot of a minor diameter of 2 .tm and a major diameter of 20 gm.

In alternative embodiments, a different shape and/or dimension(s) may beused for the beam 158. Note that other major and minor diameters areattainable by one skilled in the art and can be applied to the sameprocess.

In another embodiment, the focused energy device 157 is implemented fromthe opposite side of the interrogation beam 148, as shown in FIG. 17 .The configuration shown is advantageous because it is easily implementedand efficiently uses the free space at a photo detector side of thesystem.

In one embodiment, dichroic mirrors are used to split off specificwavelengths or to integrate specific wavelengths into the optical path.Further, although a mirror and dichroic mirror/beam splitter may beused, one of ordinary skill in the art would know that more than onemirror and/or dichroic mirror/beam splitter may be utilized in thepresent system.

In one embodiment, collection optics 153, including a microscopeobjective, collect the fluorescence emission from the objects 160 in thechip 100, and a dichroic mirror passes the fluorescence emission fromthe collection optics 153 towards the optical signal detector 154. Inone embodiment, the focused energy device 157 emits a beam 158 whichpasses through beam shaping optics 180 (as described above), and whichis directed by a mirror and also reflected by dichroic mirror throughcollection optics 153 onto the chip 100. Specifically, in oneembodiment, the objective lens of the collection optics 153 focuses thefocused energy beam 158, which enters the back aperture of the objectivelens, into a tight spot on the objects 160 just slightly downstream fromthe interrogation/excitation point in action region 129. However, one ofordinary skill in the art would know that the focused energy beam 158may be disposed below the output 112 of the chip 100, or slightlyupstream from the interrogation/excitation region 129.

In one embodiment, a distance between the beam spot of the interrogationbeam 148 and the beam spot of the focused energy beam 158, isadjustable.

In one embodiment, a dichroic mirror or any beam splitting device maysplit a small bit of light off to a camera 182 (see FIG. 5 ), allowingthe user to visually examine alignment.

In one embodiment, the camera 182 provides a visual image of themicrofluidic flow environment for general alignment purposes. The cameracan be used to determine location and timing for firing of the focusedenergy device 157.

In another embodiment, the focused energy beam 158 is implemented fromthe same side as the interrogation beam 148 (see FIG. 17 ). With thisapproach, the focusing lens for the focused energy apparatus 157 is notshared with the detection side, so it is more flexible for beam shaping,and it eliminates the need for a microscope objective that is rated forhigh power at the action (i.e., photodamage, killing) wavelength, whichcan reduce the system cost.

In this embodiment, the focused energy device 157 emits a beam 158 fromthe same side as the interrogation apparatus 147, and the beam 158 isshaped by beam shaping optics 180 (described above), to be directed andaligned by a mirror and dichroic mirror, to be focused onto the objects160 in channel 164 of the chip 100. In one embodiment, beam focusingoptics 181 (as described above), are disposed between a dichroic mirrorand the chip 100, to focus the beam 158.

As stated above, in the embodiment, the beam 158 is focused into a tightspot on the objects 160 just slightly downstream from theinterrogation/excitation point in action region 129. However, one ofordinary skill in the art would know that the focused energy beam 158may be disposed below the output 112 of the chip 100, or slightlyupstream from the interrogation/excitation region 129 (see FIGS. 6A-D).

Further, as stated above, in the embodiment, a distance between the beamspot of the interrogation beam 148 and the beam spot of the focusedenergy beam 158, is adjustable.

Object Focusing and Orientation: In conventional flow cytometry systems,since objects or cells, especially with asymmetric shapes, tend toorient as they flow close to a solid surface, the function andimprovement of object or cell orientation relies on complex nozzledesigns, such as orienting baffles or an offset structure inside thenozzle. To avoid the complex design of nozzle-based flow cytometrysystems, and their high fabrication cost, the microfluidic chip 100design of the present invention focuses, positions, and orients theobjects 160, in order to optimize its analytical capability. Thus, inone embodiment, the objects 160 (i.e., cells) with non-spherical shapesare aligned into a restricted core volume in channel 164 and maintainedin a similar and desired orientation when they pass through theinterrogation/detection beam 148. As a result, more uniform scatteringand detection signals will be obtained, thus, helping to increase thesystem's 100 sensitivity and stability.

In one embodiment, the orientation of objects 160 can be realized bypositioning the sample core stream 120 offset with respect to the centerof the central plane of the channel 164 cross-section, usinghydrodynamic focusing.

Two-Step Hydrodynamic Focusing: The following describes two-stephydrodynamic focusing that takes place during fluid flow in oneembodiment of the microfluidic chip 100 (see FIGS. 1B-1D).

In one embodiment, the first hydrodynamic focusing step of the presentinvention is accomplished by inputting a fluid sample 120 containingobjects 160, including biological samples such as sperm cells 160, etc.,through sample input 106, and inputting sheath or buffer fluids 163through sheath or buffer inputs 107, 108. In one embodiment, the objects160 are pre-stained with dye according to known methods (e.g., Hoechstdye), in order to allow fluorescence and imaging thereof.

In one embodiment, objects 160 in the sample fluid mixture 120 flowthrough main channel 164, are surrounded and shaped by the fluid flow,and have random orientation and position (see FIGS. 3A and 6A). Atintersection 161, the sample mixture 120 flowing in main channel 164 issurrounded and shaped by the sheath or buffer fluids 163 from channels116, 117, and compressed in a first direction (i.e., at leasthorizontally, on at least both sides of the flow, if not all sidesdepending on where the main channel 164 enters the intersection 161),when the sheath or buffer fluids 163 meet with the sample mixture 120.This compression is termed hydrodynamic focusing (three-dimensional(3-D)) and is used to align the objects 160 in the channel 164 into arestricted core volume that may approximate a single file formation. Thehydrodynamic focusing takes advantage of significantly large sheath orbuffer flow in channel 164 to accelerate the travelling velocity of theobjects 160 through the planar microfluidic channel 164. In oneembodiment, the sample core stream 120 may also be offset from thecentral plane by a ramp 166B or taper 166A structure in the channel 164which is prior to the junction 161 of the channel 164 and the first-stepsheath or buffer channels 116, 117.

As a result, the objects 160 are speeded up and the spacing between theobjects 160 in the microfluidic channel 164 also can be stretched. Thevelocity of the objects 160 is dependent upon the sample 120 flow rateand the ratio of the value to total sheath or buffer 163 flow rate. Thisfunction is useful to avoid clogging issues and object 160 clumping withhighly concentrated object samples 120.

However, as shown in FIG. 8A, at this stage, the resulting sample 120core stream across the main channel 164 still shows overlapped objects160 or cells along the channel 164 depth direction or vertical axis. Inparticular, the objects 160 are focused around the center of the channel164, and may be compressed into a thin strip across the depth of thechannel 164. Thus, at intersection 161, as the sample fluid 120 is beingcompressed by the sheath or buffer fluids 163 from channels 114, 115,toward the center of the channel 164, the objects 160 (i.e., spermcells) move toward the center of the channel 164 width.

In one embodiment, the present invention includes a second focusingstep, where the sample mixture 120 containing objects 160, is furthercompressed by sheath or buffer fluids 163 from a second direction (i.e.,the vertical direction, from the top and the bottom) entering fromchannels 114, 115 at intersection 162 (see FIG. 14 ). The intersection162 leading into channel 164B is the second focusing region. Note thatalthough the entrances into intersection 162 from channels 114, 115 areshown as rectangular, one of ordinary skill in the art would appreciatethat any other suitable configuration (i.e., tapered, circular) may beused.

In one embodiment, the sheath or buffer fluids 163 in the channels 114,114 enter from the same plane (see FIGS. 3A and 6A), or from differentplanes into the channel 164 (see FIG. 15 , where channels 114, 115 aredisposed above and below main channel 164, entering channel 164vertically), to align the objects 160 in the center of the channel 164Bby both width and depth (i.e., horizontally and vertically) as they flowalong channel 164B. Then, the resulting flow in the main channel 164 issubsequently compressed and repositioned by the second-step sheath flowvia microfluidic channels 114, 115.

Thus, in the second focusing step of the present invention, the samplemixture 120 is again compressed by the vertical sheath or buffer fluids163 entering at channels 114, 115, and the sample 120 stream is focusedat the center of the channel 164 depth, as illustrated in FIG. 8B, andthe objects 160 flow along the center of the channel 164 in a restrictedcore volume that may approximate a single file formation in a particularorientation.

Accordingly, after these two subsequent hydrodynamic focusing steps, arestricted core volume of objects 160 or cells is obtained and theposition of the stream also can be adjusted to a desired location alongthe vertical axis (see FIG. 8B). Thus, the objects 160 introduced intosample input 106, undergo two-step hydrodynamic focusing, which allowsthe objects 160 to move through the channel 164B in a restricted corevolume that may approximate a single file formation, in a more uniformorientation (depending on the type of objects 160), which allows foreasier interrogation of the objects 160.

Three-Step Hydrodynamic Focusing: In one embodiment, three-stephydrodynamic focusing is performed on the objects 160 in the chip 100.In this embodiment, as shown in FIGS. 1A and 3B, first two hydrodynamicfocusing steps are accomplished by horizontally compressing the samplefluid stream 120 at intersections 161 and 162, and then in a third step,the sample fluid stream 120 is vertically compressed in the channel 164.Sheath or buffer channels 114, 115, and 116, 117 enter the channel 164from a horizontal direction, at an angle of 45 degrees or less for eachchannel.

More specifically, in the first hydrodynamic focusing step, sample 120flow enters into the first intersection 161, and the sheath or bufferfluids 163 from channels 116, 117 surround the sample 120 flow andimmediately compress it into a thin sample 120 stream in channel 164. Inone embodiment, the sample fluid channel 164 is tapered with an internalramp prior to intersection 161, where sheath or buffer fluid channel116, 117 enter the channel 164 (see FIG. 3B). Meanwhile, as the channel164 is shallower (i.e., smaller in dimension) than the first sheathchannels 116, 117, the sample 120 stream is lifted by the sheath orbuffer fluid 163 from channels 116, 117, to flow to the top of the mainchannel 164.

In the second hydrodynamic focusing step, the sheath or buffer fluids163 are introduced from channels 114, 115 into channel 164—the channel114, 115 which are disposed close to the top of the main channel 164(see FIG. 3B). In one embodiment, as shown in FIG. 33 , sheath or bufferchannels 114, 115 join channel 164 horizontally, and may be of a smallerdimension from that of sheath or buffer channels 116, 117. Thus, sincethe depth of the channels 114, 115 are shallower than the main channel164, the sheath or buffer fluids 163 further compress the sample 120stream along the channel 164 width, to constrain the width of sample 120stream. This sheath or buffer fluid 163 flow significantly improves thesignal measurement sensitivity.

Without a third hydrodynamic focusing step, the lack of verticalcompressing at intersections 161 and 162 may result in multiple objects160 or cells simultaneously entering the detection region 129, thus,reducing the detection sensitivity or causing measurement errors,especially for a high throughput flow cytometry application.

However, three-dimensional hydrodynamic focusing is an effective way toalign the objects 160 in a restricted core volume that may approximate asingle file formation in channel 164. Further, the consistentpositioning of the object 160 in the channel 164 results in minimumvariability in velocity from object to object in the parabolic flow.

In microfluidic-based flow cytometry, the flow velocity profile isparabolic along the rectangular cross-section of the micro-channel. FIG.7 illustrates the profile of shear force on the cross-section of themicrofluidic channel 164. The shear force gradient shown in FIG. 7 hasminimum shear force at the tapered tip and the larger region at the rearhas the maximum shear value. Thus, the hydrodynamic shear force gradientis formed across the channel 164 cross-section. The large shear forceclose to the channel 164 wall helps to orient asymmetric objects 160.

In the present embodiment, after the first two steps of hydrodynamicfocusing, the sample 120 flow shrinks into a very thin stream in bothhorizontal and vertical directions. The ratio of independentlycontrolled sheath or buffer fluids 163 from channels 116, 117 and 114,115, each determines the size of the resulting sample 120 stream inchannel 164. After the compression caused by sheath or buffer fluid 163from channels 114, 115, the objects 160 may spread a little along thechannel 164 depth, but objects 160 still follow the sample 120 stream,which is very close to the top ceiling of the main channel 164 (note:with the chip described as horizontally disposed for ease of reference).Therefore, it is necessary to have a third sheath or buffer fluid 163flow from channel 172 to further compress the sample 120 stream alongthe vertical direction, and confine the objects 160 well inside thethin, sample 120 stream.

Additionally, since the sheath or buffer fluid 163 flow is introducedperpendicularly to the pre-confined sample 120 stream from sheath orbuffer channel 172, it helps to position the sample 120 stream to alocation along the cross-section of the channel 164 (i.e., achieves anend result as shown in FIG. 8B). By fine-turning the flow rate of sheathor buffer fluid 163 from channel 172, the objects' 160 positions can beprecisely controlled when they pass through the detection region 129.

In one embodiment, the sheath or buffer fluid 163 is introduced bychannel 172 from external sheath tubing (see FIG. 20 ) instead of bymicro-channels running through the microfluidic chip 100. Thus, anexternal flow controller is required to provide a constant and stableflow rate through the input channel 172 (see FIGS. 19-21 ).

The design of the present invention allows the core sample stream 120 toorient flat-shaped objects, position the objects 160 in the channel 164in a physical arrangement approaching uniformity, all of which improvesthe downstream precision action of the focused energy apparatus 157.

Although three hydrodynamic focusing steps are disclosed above, one ofordinary skill in the art would know that the configuration and numberof the sheath or buffer channels may change, as long as they achieve thedesired features of the present invention, with respect to theorientation and focusing of the objects in the sample fluid 120.

Flow control methods: To realize the exemplary three-dimensionalhydrodynamic focusing methods described above, both sample fluid 120 andsheath or buffer fluids 163 are required to be precisely delivered sothat a constant flow can be streamed through the microfluidic chip 100.After being compressed by the sheath or buffer fluid flows 163, theobjects 160 or cells have been accelerated and the average spacingbetween the objects 160 or cells in the sample 120 core stream is alsostretched significantly therefrom. The ratio of the total sheath orbuffer fluid 163 flow rate and the sample 120 flow rate can be adjustedbetween 100:1 and 1000:1. Preferably, the ratio of 200-400:1 is used inthe microfluidic chip 100 of the present invention. The overall fluidflow rate in the microfluidic chip 100 is about 2-4 ml/min. Theintroduced sheath or buffer fluid flows 163 have to be constant andpulse-free to ensure a stable traveling speed of the objects 160 duringinterrogation and signal detection, and between thedetection/interrogation position and the position of the action of thefocused energy apparatus 157 (see FIG. 6A). This facilitates an accuratesignal reading and action on the target object 160 by the focused energyapparatus 157. With the precise control of fluid flow through the mainchannel 164, the overall flow rate variation is less than 1% of the setflow rate, and the traveling speed of target objects 160 for potentialaction by the focused energy apparatus 157 varies less than 1% from theposition where interrogation and detection of objects 160 takes place,to the position where the focused energy apparatus 157 acts on theobjects 160 (see FIG. 6A).

Orientation of Objects: One of the challenging issues in the detectionof flat-shaped objects 160 (i.e., sperm cells) is to constrain theobjects 160 in a uniform orientation when passing through theinterrogation beam 148. Thus, an approximately uniform positioning ofobjects 160 and a corresponding orientation of objects 160 in thechannel 164, helps to increase the sensitivity of the system. With theaforementioned hydrodynamic focusing strategy, the position of objects160 along the channel 164 can be manipulated in a controlled way. Thus,by adjusting the ratio between the sheath or buffer fluid 163 flows fromchannels 116, 117, and 114, 115, and 172, a position of the focusedsample 120 stream offset from the center of the channel 164 by about5-20 microns (based on a channel 164 cross-section of 150-micron width;and 100-micron heights, for example), is preferred for the detection offlat-shaped objects 160. Generally, an adjustment of 0-100 microns biasposition of the objects 160 can be achieved.

Specifically; in order to align objects 160 in the channel 164 toimprove their orientation, the high aspect ratio of the microfluidicchannel 164 is taken advantage of to induce the shear force to turn theflat surface of the object 160 (i.e., sperm cell) facing the channel 164wall. Further, the sheath or buffer fluid 163 flow can be activelyemployed to compress and position the objects 160 in the channel 164.These methods are described below in more detail.

Passive Method: In one embodiment, an asymmetric geometry structure maybe utilized to position the focused objects 160 in the channel 164, byone of: a) placing an asymmetric ramp 166B in the sample main channel164 to lift the sample flow 120 (see explanation above regarding FIG.4A); and b) placing an asymmetric ramp 166B in the main channel 164prior to the action chamber 129 to lift up the focused sample stream 120(see explanation above regarding FIG. 4B). The above asymmetric featurescan be used individually or in an appropriate combination of two ormore. However, one of ordinary skill in the art would know that thesefeatures are not necessary to the achievement of the position of theobjects 160 in the channel 164.

In one embodiment, as noted above, ramps may be used in the channels114-117 to lift the sample flow 120, although they are not necessary.The placement of ramps in the channels is dependent upon the directionin which the objects 160 of the sample core stream 120 are required tobe offset from the center to improve object 160 or cell orientation.However, the above passive method has less flexibility to vary theobject 160 position in the main channel 164.

Active Method: In an alternative method to offset the sample 120 corestream in channel 164, an asymmetric sheath flow 163 is introduced toadjust the positions of objects 160 or cells in the channel 164. Thereare several methods of realizing asymmetric sheath or buffer fluid flow163, two embodiments of which are described below.

One embodiment is to introduce a single sheath or buffer fluid flow 163which forms a 90 degree angle with the main channel 164 wall, as shownin FIG. 3A (in a two-step hydrodynamic focusing method) or FIG. 3B (in athree-step hydrodynamic focusing method). In the two-step hydrodynamicfocusing embodiment, the introduced sheath or buffer fluid flow 163 atthe second-step hydrodynamic focusing intersection 162, furthercompresses the sample core stream 120 subsequent to the first-stephydrodynamic focusing at intersection 161.

In the alternative three-step hydrodynamic focusing embodiment, thiscompressing of the sample core stream 120 occurs at the third-stephydrodynamic focusing intersection where channel 172 joins main channel164. Thus, the final hydrodynamic focusing step positions the objects160 or cells to a desired location along the vertical axis. Bycontrolling the ratio of the hydrodynamic focusing flow rates, a desiredposition of the objects 160 can be obtained to achieve the optimumorientation.

In a second embodiment using the two-step hydrodynamic focusing method,two second-step sheath or buffer channels 114, 115, merge in an angle tothe main channel 164 wall, and parallel main channel 164 from above andbelow, as shown in FIGS. 12A-12B. The angle of the second-stephydrodynamic focusing channels 114, 115, and main channel 164 may varyand is dependent upon the fabrication methods. Preferably, a 90-degreeangle is selected (see FIGS. 12A-12B). Different flow rates of sheath orbuffer fluids 164 may flow via the two channels 114, 115, which arecapable of repositioning the sample core stream 120 in the main channel164. After the objects 160 are offset from the central plane of thechannel 164, orientation of the objects 160 is improved. In a specificembodiment such as sperm cells 160, the sperm cells 160 tend to turntheir flat sides to the channel 164 wall in the vertical axis.

As can be seen from the embodiment of FIG. 12B, the dimensions of thechannels 114, 115 are not necessarily identical (as shown in FIG. 12A),in order to obtain different hydraulic resistances. Thus, the same flowrate of the second-step sheath or buffer fluid flows 164 in the channels114, 115 will also generate bias fluid flow as well.

To summarize, both of the passive and active methods above can help tooptimally position the objects 160 and improve their orientation in thechannel 164.

In one embodiment, pancake-shaped sperm cells 160 are taken as anexample of the objects 160. Because of their pancake-type or flattenedteardrop-shaped heads, the sperm cells 160 will re-orient themselves ina predetermined direction as they undergo the second, or third(depending on the embodiment) focusing step—i.e., with their flatsurfaces perpendicular to the direction of light beam 148 (see FIG. 6 ).Thus, the sperm cells 160 develop a preference on their body orientationwhile passing through the hydrodynamic focusing process. Specifically,the sperm cells 160 tend to be more stable with their flat bodiesperpendicular to the direction of the compression. Hence, with thecontrol of the sheath or buffer fluids 163, the sperm cells 160 whichstart with random orientation, now achieve uniform orientation. Thus,the sperm cells 160 are not only disposed in a restricted core volume atthe center of the channel 164B, but they also achieve a uniformorientation with their flat surface normal to the direction ofcompression in the last hydrodynamic focusing step.

The above methods improve the sperm cells' 160 orientation and thecapability to differentiate the DNA content of X- and Y-spermchromosomes (and thereby distinguish between X and Y sperm). Thehistograms of FIGS. 13A and 13B show the sperm cells 160 in the centerof the channel 164, and offset from the center of the channel 164,respectively. The circles on the left and right of the histogram in FIG.13A shows the populations of mal-orientated sperm cells 160. The lefthump of the major population weakens the capability to differentiate X-and Y-sperm populations and contributes to the asymmetric distributionof X- and Y-sperm populations.

Operation of Microfluidic Chip System

Interrogation of Objects: In one embodiment, the interrogation lightsource 147 is an excitation laser 147 (see FIG. 16 ), having 350 mWpower, 355 nm wavelength, 12 ps pulse width.

In one embodiment, further downstream from the hydrodynamic focusingsteps, in channel 164, the objects 160 are detected in the actionchamber 129 at opening 150 through covering 133, using the light source147. Light source 147 emits a light beam 148 (which may be via anoptical fiber) which is focused at the center of the channel 164 atopening 150.

In one embodiment, the objects 160 are sperm cells 160, which areoriented by the hydrodynamic focusing steps, such that the flat surfacesof the sperm cells 160 are facing toward the light beam 148. Inaddition, all objects 160 or sperm cells 160 are moved into a restrictedcore volume that may approximate a single file formation, by thehydrodynamic focusing steps, as they pass under light beam 148. As theobjects 160 pass under light source 147 and are acted upon by light beam148, the objects 160 emit the fluorescence which indicates the identityof the desired objects 160.

The light source 147 provides the fluorescence excitation energy fordetection of objects 160 in the action region 129. In one exemplaryembodiment with respect to the objects 160 being sperm cells 160, Xchromosome cells fluoresce at a different intensity from Y chromosomecells (based on DNA content, as is well known in the art) (note: 355 nmis selected for the Hoescht 33342 dye used on the DNA). Further, inother embodiments, objects 160 which are cells carrying one trait mayfluoresce in a different intensity or wavelength from cells carrying adifferent set of traits. In addition, the objects 160 can be viewed forshape, size, or any other distinguishing indicators.

Thus, in the embodiment of sperm cells 160, the illumination to the flatsurface and the edge of the cells 160 is quite different with spermcells 160 as compared to other cells. The fluorescence signal derivedfrom the edge of the sperm cells 160 is significantly stronger than thatfrom the flat surface, which increases the difficulty for the digitalprocessor 156 to deal with the stronger signal from the edge, and thenormal lower signal from the X- and Y-flat surface of the cells 160.Thus, turning the flat surface of the sperm cell 160 to face the laserillumination (i.e., light beam 148) helps to reduce the orientationvariability and increase the capability of the system to differentiateX- or Y-sperm cells 160.

In the embodiment of beam-induced fluorescence, the emitted light beam151 (in FIG. 5 ) is then collected by the objective lens 153, andsubsequently converted to an electronic signal by the optical sensor154. The electronic signal is then digitized by an analog-digitalconverter (ADC) 155 and sent to an electronic controller 156 for signalprocessing.

As noted above, in one embodiment, the DSP-based controller 156 monitorsthe electronic signal, and when a particular signal is noted, a focusedenergy apparatus 157 may be employed to act upon a target object 160(see FIGS. 6A-6C). However, in an alternative embodiment, theinterrogation apparatus interrogates the objects 160 after they areacted upon by the focused energy device 157 (see FIG. 6D).

In one embodiment, interrogation of the sample 120 containing objects160 (i.e., biological material), is accomplished by other methods. Thus,portions of, or outputs from, the microfluidic chip 100 may be inspectedoptically or visually. Overall, interrogation methods may include directvisual imaging, such as with a camera, and may utilize directbright-light imaging or fluorescent imaging; or, more sophisticatedtechniques may be used such as spectroscopy, transmission spectroscopy,spectral imaging, or scattering such as dynamic light scattering ordiffusive wave spectroscopy.

In some cases, the optical interrogation region 129 may be used inconjunction with additives, such as chemicals that bind to or affectobjects 160 of the sample mixture 120, or beads which are functionalizedto bind and/or fluoresce in the presence of certain materials ordiseases. These techniques may be used to measure cell concentrations,to detect disease, or to detect other parameters which characterize theobjects 160.

However, in another embodiment, if fluorescence is not used, thenpolarized light back scattering methods may also be used. Usingspectroscopic methods, the objects 160 are interrogated as describedabove. The spectrum of those objects 160 which had positive results andfluorescence (i.e., those objects 160 which reacted with a label) areidentified for selection by the focused energy apparatus 157.

In one embodiment, the objects 160 may be interrogated and identifiedbased on the reaction or binding of the objects 160 with additives orsheath or buffer fluids 163, or by using the natural fluorescence of theobjects 160, or the fluorescence of a substance associated with theobject 160, as an identity tag or background tag, or meet a selectedsize, dimension, or surface feature, etc.

In one embodiment, upon completion of an assay, selection may be made,via computer 182 (which monitors the electronic signal and employs thefocused energy apparatus 157) and/or operator, of which objects 160 todiscard and which to collect.

Applications for Focused Energy Apparatus: The focused energy apparatus157 of the present invention may carry out a number of actions on theobjects 160 in channel 164, or between chip 100 and container 188.

In one embodiment, the focused energy device 157 acts to photodamage ordestroy the objects 160 in a number of ways.

Specifically, the focused energy apparatus 157 acts to kill objects 160(i.e., cells). For example, the target objects 160 may be unwanted cells160, and upon action by the focused energy apparatus 167, cellular deathmay be caused by overheating of the intracellular environment, which maypromote, but is not limited to, protein denaturation or reduction inenzyme activity.

In another method, the action of the energy dosage 158 from the focusedenergy apparatus 157 is strong enough to cause rupture of the plasmamembrane and leaking of the cellular contents out of the cell 160 andinto the surrounding environment (i.e., sheath or buffer fluid 163).

In another method, object 160 or cell death can be caused by theformation of radical oxygen species (ROS) due to adsorption of energyfrom the focused energy pulses 158 from the focused energy apparatus157, which will cause, among other things, DNA and protein damage.

In another embodiment, the focused energy apparatus 157 can temporarilyor permanently disable target objects 160, such as cells 160, usingfocused energy pulses 158 from the focused energy apparatus 157.

For example, exposing sperm cells 160 to focused energy pulses 158 suchas those produced by a laser or LED 157 generates photo-activationwithin the cells 160 and results in temporary or permanent disablementof cellular mechanisms responsible for sperm 160 motility. Afterdisabling target sperm cells 160, the resulting sample 120 containsmotile sperm 160 and immotile (target) sperm 160, where the immotilesperm are unable to fertilize oocytes naturally.

In another embodiment, it may be desirable to use focused energy pulses158 to make sperm 160 infertile through the dimerization of nucleotidesin the DNA. Dimerization occurs when cells 160, such as sperm cells 160,are exposed to UV light, causing bonds between pyrimidine bases, andresulting in a type of “cross-linking”, which, if not repaired, inhibitsreplication and transcription. Thus, although the target sperm cells 160are still alive as evidenced by their motility, the fertility of thetargeted sperm cells 160 is greatly reduced.

In addition to sperm cells 160, one can use high-powered focused energysources 157 such as LEDs or lasers 157 to photobleach fluorescence inobjects 160, such as cells or colloids 160, which express apredetermined level of fluorescence. For instance, in many self-assemblyobject formulations, a wide size range of objects 160 are formed thatare difficult to separate from each other. To produce an enhanced sample120 of objects 160 having a desired size, one can fluorescently labelall objects 160 using methods known in the art, and one can use opticalinterrogation to determine object 160 size, and photobleach objects 160possessing the predetermined level of fluorescence.

In a specific example, a semen sample 120 may contain contaminants suchas bacterial or viral cells, which are the target of photobleaching.Another example may include sperm cells 160 containing a given trait ofthe cell 160 or DNA and labeled with the fluorophore for quantitativeand/or qualitative measurement. Sperm cells 160 containing the trait maybe targeted by the focused energy apparatus 157. In another embodiment,sperm cells 160 which do not contain the trait may be targeted forphotobleaching. Specific cells 160 in other cell mixtures such as bloodare also candidates for photobleaching treatments to reduce viability.In yet another embodiment, photobleached cells/objects 160 can beundetectable downstream and therefore, are not subject to subsequentprocessing steps (i.e., can bypass subsequent processing steps).

In another embodiment, in contrast to the disabling of objects 160,focused energy pulses 158 can be used to activate materials such ascaged molecules or compounds within the objects 160.

In one application, the caged compounds represent but are not limitedto, fluorescent markers or cell responsive molecules. In theseapplications, focused energy pulses 158 are used to causephoto-activation of the caged molecules or compounds which alters cell160 signaling kinetics for ex-vivo therapies.

In another embodiment, focused energy pulses 158 can activatephoto-polymerization events which disable cells or colloids 160 byaltering internal properties of the object 160.

In another embodiment, with respect to intracellular signaling pathways,focused energy pulses 158 are used to activate heat shock proteins orinduce mitochondrial biogenesis or activation within objects 160 orcells, including germ cells, to enhance cellular viability andfunctionality. Additional intracellular pathways may also be activatedto repair damage done by either the interrogation/detection device 147or a number of other factors that are too numerous to list (i.e.,environmental, heat, chemical, etc.)

In one embodiment, one skilled in the art can generatephoto-polymerization through focused energy pulses 158 to temporarilyencapsulate or permanently contain target cells 160, colloids, or otherobjects, using multi-armed PEG-acrylate/PEG-vinyl/etc., which promotesencapsulation of the target objects 160 or cells. Sperm cells 160 can beencapsulated to enhance the preservation of viability and fertilitythrough commercial storage and delivery processes.

In another embodiment, focused energy pulses 158 are used to causephoto-polymerization events on the surface of targeted cells or objects160 which increase the size or density of the encapsulating material inorder to alter the size or density of the target object 160 or improveproperties and performance of the encapsulating material.

In another embodiment, a photopolymerizable sequence in the hydrophobicportion of the vesicle may be used to permanently seal the desiredmolecule or object 160 by encapsulation therein.

In another method, the focused energy apparatus 157 may be used to acton externally or alter the environment around the target objects 160.

In one method, focused energy pulses 158 are used to heat the localenvironment around target cells or objects 160 so that the thermalenhancement is sufficient to cause toxicity to target cells 160.

In another embodiment, focused energy pulses 158 are used to promoterupture of analytes containing delivery vehicles (such as vesicles)which are in close proximity to target cells 160. The delivery vehiclescarry molecules such as sodium fluoride (NaF) which causes temporaryimmobility of sperm cells 160, or heparin which promotes capacitation ofspermatozoa 160. When the concentration of analytes or activating agentsare increased locally, target sperm cells 160 or other objects respondto the local signals without activating similar responses in non-targetcells 160.

In another embodiment, when objects 160 or cells are attached to asurface, the surrounding environment is modified with focused energypulses 158 to vary the modulus of elasticity of the surface or releasecell responsive chemicals from the surface of surrounding objects 160 orcells.

In one embodiment, heat production through absorption of light/EM waves158 causes a temperature change which kills the objects 160 or cells.

In another embodiment, focused energy pulses 158 are used to formpredetermined chemical bonds or break chemical bonds in the attachmentmaterial, thus, directing the differentiation of objects 160, such asstem cells 160, into differentiated cell lines.

In one embodiment, for some applications, it is desirable to use focusedenergy pulses 158 to promote cellular uptake or adhesion onto targetobjects 160.

In one embodiment, cellular uptake of antibodies, cellular probes, orDNA is enhanced through local heating.

In one embodiment, with said local heating, when the temperatureincrease is optimized also to maintain object 160 or cell viability, theinternalization of objects 160 into target viable cells 160 isselectively promoted.

In one embodiment, the object 160 to be delivered is attached to anobject that when targeted with a light source 147, may cause a briefmicrobubble. For instance, an oligonucleotide may be conjugated to agold nanoparticle. When the gold is heated with a light source 147 at anoptimized wavelength, a microbubble is briefly formed. Upon cavitationof the microbubble, the gold nanoparticles are broken apart and thepieces of the nanoparticle and the object attached to it arepermeabilized through a cell membrane.

In another embodiment, the temperature increase of the target object 160is not sufficient to cause the formation of a microbubble. The localizedtemperature increase is optimized to maintain cell viability andselectively promote the internalization of objects 160 into targetviable cells.

Similarly, in another embodiment, by using focused energy pulses 158 toadhere materials onto colloids or objects, object 160 geometry or object160 properties is altered, thus enabling additional separationtechniques, such as magnetic or electric fields to separate materialsthat are normally not susceptible to such forces.

Operation of Focused Energy Apparatus: Generally, flow cytometricanalysis and action systems that use an electromagnetic radiation sourcesuch as a focused energy apparatus 157 or laser, to act on selectedobjects 160, typically desire to deliver a controlled energy level toindividual objects 160. In one embodiment, such systems can kill, alter,damage, or destroy targeted objects 160 or cells using the focusedenergy apparatus 157. In other embodiments, such systems can, amongother methods, activate targets in selected objects 160 or cells or inthe fluid, media, or matrix surrounding selected objects 160, asdescribed above.

In the above methods which utilize focused energy pulses 158, radiationcan be applied by methods of either targeted firing or continuouselimination, such that the desired objects 160 are unaffected, and theunwanted, altered, killed, destroyed, or damaged objects 160 arediscriminated from the sample 120. Similar considerations as notedabove, are given when selecting laser wavelength and laser power fortargeted firing and continuous firing modes.

Targeted Firing: More specifically, in targeted firing, the focusedenergy apparatus 157 is employed for targeted objects 160. Specifically,an object 160 in a sample 120 fluid mixture, may pass through aninterrogation/detection area in chamber 129, for example, wherespecified characteristics of the object 160 are evaluated by one or moreof the above methods.

Thus, in a flow-based system, for example, the focused energy apparatus157 action area 129 is downstream from the optical interrogation areausing light source 147 for interrogation. Alternatively, the focusedenergy apparatus 157 is utilized prior to interrogation furtherdownstream. In one embodiment, the focused energy apparatus 157 acts onthe objects in action chamber 129. The distance between the opticalinterrogation region and the action region may be adjusted toaccommodate different timings.

Based upon predetermined criteria, a decision is made to either keep,discard, or act upon the selected object 160. Objects 160 marked foraction are hit with a triggered pulse of energy 158 from the focusedenergy apparatus 157 (see FIGS. 6A-60 , for example).

When the object 160 or cell is not targeted, it remains unaffected, andflows through the chamber 129, via channel 164 to output channel 141 andcontainer 188, which collects target and non-target objects 160 as adiscriminated product 165.

In one embodiment, the laser pulse 158 has a short duration and canselectively target individual objects 160 or cells while exerting nointended impact on non-target objects 160 or cells which may be nearby,thus, avoiding “overspray” to non-target objects 160 or cells. Pulseenergy is selected to impart the desired effect while avoiding undesireddisturbances to the surrounding media, or for example, in flow systems,does not cause unintended cavitation or bubble formation. A variety oflaser wavelengths can be used; however, the flux requirement may bedifferent depending on the characteristics of the target object 160,dye, and environment.

Laser units 157 have limited power, especially those compact models thatoperate at high pulse frequency (>100 kHz typically), and it may bepreferable to choose a laser wavelength that minimizes the requiredflux. For example, matching the laser 157 wavelength to the absorbanceof dyes, other targets, or objects 160 (i.e., molecules) used in theaction process greatly improves efficiency and effectiveness.Additionally, pulse energy 158 is selected to impart the desired effectwhile avoiding undesired disturbances to the surrounding media, or forexample, in flow systems, not causing unintended cavitation or bubbleformation.

In one specific example related to sperm cells 160 as the objects 160, a355 nm laser 157 was used to take advantage of the dye (i.e., Hoechst33342 dye) used for the cell staining process. In a similar example, a349 nm laser 157 may be used. In such examples, when unwanted spermcells 160 are photodamaged, destroyed or killed, pulse energy levels of0.5-8.0 pJ are used. FIG. 26 shows the excitation wavelength and theemission wavelength for cells stained with Hoescht 33342.

Continuous Firing: In continuous firing, such as in a flow-based system,the focused energy pulse 158 is constantly employed and only interruptedfor the passage of the non-target objects 160 (i.e., wanted objects 160that are not to be damaged, destroyed, altered, or killed), or debris orcontaminants which do not require action thereon.

As stated above, based upon predetermined criteria, a decision is madeto either keep or discard an object 160, or act upon, includingphotodamage, kill, alter, disable, or destroy an object 160. The focusedenergy apparatus 157, such as a continuous wave (CW) or rapidly pulsedlaser or LED, delivers a continuous stream of focused energy 158 to theobjects 160, and is used to act upon (i.e., including, photodamage,alter, disable, destroy, or kill) every object 160 passing through aparticular location in the flow stream of the sample 120 fluid flow inan exemplary flow system. When non-target objects 160 are encountered inthe action region, the laser beam 158 is shut off, deflected, orotherwise interrupted for a short period of time, to allow thenon-target objects 160 (in some cases, discarded items), to pass throughunaffected. The objects 160—target or non-target flow through channel141 into container 188.

Methods for interrupting or diffusing the beam 158 include mechanical(shutters, choppers, galvanometer mirror), optical (acoustic opticdeflector, acoustic optic modulator, spatial light modulator, digitalmicromirror device, polarization modification, liquid crystal display),electronic (pulse conditioning, dropping, or alteration of a Q-switchlaser), or acoustic. Any other known or future suitable methods ortechniques may be utilized to interrupt the focused energy beam 158.

With either targeted or continuous methods, the energy pulse 158 has ashort duration and the focused energy apparatus 157 can selectivelytarget only a single object 160 and not impact other objects 160 whichare nearby in the sample 120 fluid flow (i.e., can limit “collateraldamage”). The energy pulse 158 is selected to be sufficient to achievethe desired action on the object 160 (i.e., damage, alter, kill ordestroy the object 160), per user requirements. The pulsed energy 158from the focused energy apparatus 157 should fall within a range whereit will not cause a disturbance to the sample 120 fluid due tocavitation, bubble formation, or method of energy absorbance.

Other technologies for reducing unintended action, damage, anddestruction to non-target objects 160 include those which absorb asignificant portion of the pulse energy 158, alter the direction of thebeam 158, or discharge excess energy by firing pulses 158 into the flowstream 120. Specifically, these can include mechanically moving a mirroror lens so as to defocus or deflect excess laser pulses 158 into anenergy absorptive device, lenses altered electronically in order tochange the laser's 157 propagation angle, and sophisticated triggeringtechnologies which coordinate pulse energy data from the laser 157 withdata about the object-to-object timing of objects 160 in the immediateflow stream.

Pulse Timing: The timing between actions by the focused energy device157 on the objects 160 is not uniform and follows a Poisson distributionwhere many short and extremely long intervals occur. Because alaser-based action system 157 includes inherent limiting factors, it ispreferable to include a short “recharge time” between laser pulses 158.The latency time (inherent in the focused energy apparatus 157), plusthe “charge/recharge” time, is the minimum time that the focused energyapparatus 157 can react (deliver a pulse) and still provide the requiredenergy level to the targeted object 160.

In one embodiment, charge time should range from 0.1 Its to 1 second,and preferably should be from 0.1 μs to 4 ms. Pulse-to-pulse variabilityin energy levels affects the rate of producing the desired effect ontarget objects 160 or cells, and the potential for impacting non-targetobjects 160 or cells. When fired at non-uniform intervals,pulse-to-pulse stability should be high. In one example, a Q-switchlaser 157 in pulse-on-demand mode was used to deliver average pulseenergy of 1.8 μJ with a range for individual pulses of 1.3 μJ to 2.3 μJ.

In a flow based system, the action region 129 may be located downstreamfrom the optical interrogation region in the chamber 129, or upstreamthereof, and the distance between the optical interrogation region andthe action region may be adjusted to accommodate different timings. Toaccommodate sufficient change time for a pulsed laser 157, the minimumtiming between interrogation of the objects 160 or cells, and action onselected objects 160 or cells, should be no less than 1 μs.

The focused energy apparatus 157 operates successfully, for substantialperiods, at action rates up to 5,600 objects per second, with accuracyrates which can be selected, and range for example, from 75-95%. Insystems where spacing between objects 160 in the flow stream iscontrolled, the system 157 can operate at action rates up to therepetition rate of the laser 157.

Selection of Objects: In one embodiment, the focused energy apparatus157 is employed prior to interrogation of the object 160. However, inanother embodiment, in order to determine which objects 160 are selectedfor action by the focused energy apparatus 157, as noted above, ahistogram, or any graphical representation of the measured/calculatedcharacteristics of the objects 160 after interrogation, can be used inorder to make the decision for action thereof on the population ofobjects 160. In one embodiment, after interrogation is accomplished, theplot of the span (i.e., transit time through the interrogation region ofthe chamber 129) can reflect the relative size of the sample 120 corestream under different flow conditions, object 160 or cell distributionacross the main channel 164, and object 160 traveling velocity, as wellas the variation of object 160 velocity within a particular chip 100design.

As noted above, the high aspect ratio of the main channel 164 isimportant to object 160 or cell hydrodynamic focusing, migration, andorientation. In one embodiment, less than one for the high aspect ratiofor the microfluidic channel 164 is used in the present invention.Preferably, ⅔ high aspect ratio is used for the main channel 164. Thevalue of the span itself roughly indicates the object 160 velocity.Large span value indicates that objects 160 pass the interrogation lightbeam 148 slowly. The tight size of the span indicates that the sample120 core stream is closer to the channel 164 central plane and there isless object 160 velocity variation.

In one embodiment, to precisely act on the selected objects 160 whetherflowing through chip 100 or departing from output 112, less velocityvariation of objects 160 is allowed to ensure that the focused energyapparatus 157 can precisely target the selected objects 160 or cells.Thus, based on the above-described positioning and orientation methods(i.e., active, passive methods), the objects 160 are positioned close tothe center of the cross-section of the channel 164 to reduce thevelocity variation of the objects 160.

In one embodiment, for flat-shaped object 160 or cells, such as livesperm cells 160, both orientation and velocity variation need to betaken into consideration. Thus, sperm cells 160 pushed offset from thecentral plane of the channel 164 along the vertical axis (see FIG. 9B),tend to obtain a better resolution (e.g., the differentiation of X- andY-sperm cells 160 is more than 50% separated on the histogram obtainedafter interrogation), and a less mal-oriented cell 160 population. Thus,resolution and target (i.e., photodamage, killing) efficiency arebalanced, with, in one exemplary embodiment, the sample 120 core streambeing preferably shaped into about 10 microns in width, and 5-10 micronsin height of the main channel 164 across the cross-section of the mainchannel 164 in the interrogation/detection region of chamber 129, andoffset by about 2-10 microns from the central plane of the main channel164, preferably by controlling the hydrodynamic focusing steps.

In one embodiment, where the objects 160 are sperm cells, a target spermcell 160 may be a male-bearing sperm cell (i.e., a Y chromosome-bearingsperm cell) and a non-target sperm cell 160 may be a female-bearingsperm cell (i.e., an X chromosome-bearing sperm cell). In anotherembodiment, a target sperm cell 160 may be a female-bearing sperm cell(i.e., an X chromosome-bearing sperm cell) and a non-target sperm cell160 may be a male-bearing sperm cell (i.e., a Y chromosome-bearing spermcell).

In one embodiment, the objects 160 are acted upon prior tointerrogation, by utilizing localized heat shock to incorporatemolecules such as DNA or other probes through protective outer layersand into objects—i.e., through protective membranes and into cells (seeFIG. 6D). Traditional methods for incorporating molecules using the highvoltages requires for successful electroporation to permeabilizemembranes may be desirable, as evidenced by the high cell mortalityrates. Localized heat shock represents a more gentle procedure andtherefore, may be more desirable for maintaining viability of the cells.The focused energy apparatus 157, is used to generate a localized risein temperature, thus achieving heat shock. The localized heat shockresults in permeabilizing the objects 160, thus, facilitatingincorporation of the desired molecules. An interrogation apparatus 147is used thereafter, to detect and interrogate the objects 160 from whichthe interrogation apparatus 147 determines the number or proportion ofobjects 160 for which incorporation of the molecules has been attained.This method may be particularly desirable in biodetection, cellularengineering, targeted therapeutics, and drug/gene delivery.

Action Zone: In one embodiment; after interrogation is performed; andafter an acceptable histogram is obtained (i.e., with acceptableresolution and relatively small span distribution), then the decision ismade to employ the focused energy apparatus 157 to act on the selectedobjects 160 or cells. One of the more important parameters is the timingsetting for the pulse from the focused energy apparatus 157 (i.e., delayor time interval for the object 160 between the interrogation/detectionbeam 148 and the focused energy beam 158).

The focused energy apparatus 157 action zone is the area in thecross-section of the main channel 164 where the selected objects 160 orcells can be effectively acted upon (i.e., photodamaged, altered,disabled, killed, destroyed, etc.), as shown in FIG. 16 . Based on apredetermined energy level and beam shape of the focused energyapparatus 157, and on the microfluidic channel 164 design and flowconditions; the action zone can be estimated by an action percentage(e.g., <97%). The energy level of the focused energy apparatus 157 isdependent upon the current and charging time of the focused energyapparatus 157. The larger overall flow rate in the main channel 164means that the objects 160 are traveling faster. Thus, the transit timeof the objects 160 through the focused energy beam 158 is shorter. Forexample, for an energy level of the focused energy apparatus 157 of 2.3tJ with a certain beam shape (e.g., 2.5 microns×15 microns), and object160 traveling velocity around 7.5 m/s, the focused energy apparatus 157action zone is estimated to be about 20 microns in the Y-axis direction,and about 16 microns in the vertical direction.

Within the action zone, the percentage of affected (i.e., damaged,altered, or killed) target objects 160 also relies on the shape and theposition of the sample 120 core stream. By adjusting the flow rates ofthe sheath or buffer hydrodynamic focusing flows, the size and positionof the sample 120 core stream can be tailored to accommodate the actionzone in the horizontal and vertical directions. Eventually, at thedesired energy level of the focused energy apparatus 157, flowconditions for the present microfluidic chip 100 can be determined.Thus, the shape of the sample 120 stream can be preferably constrainedto around an exemplary 10 microns in width and 5-10 microns in height.

In other embodiments, the action zone as substantially described aboveis disposed prior to interrogation, or after the sample fluid 120 leavesthe chip 100 for container 188.

Operation on Sperm Cells: As discussed above, in one exemplaryembodiment using sperm cells 160 as the objects 160, the live spermcells 160 (i.e., bovine sperm cells, with approximately 50-50X-chromosome and Y-chromosome cells) are introduced into sample input106, and pass through hydrodynamic focusing steps, to reach theinterrogation region 129. In the interrogation region 129, the dye(i.e., Hoechst 33342 dye) is excited by an interrogation beam 148 from alight source 147, such as a laser 147, which generates fluorescence inthe cells 160 which is captured by an optical signal detector 154 afterpassing through an objective lens 153.

Based on characteristics of the fluorescence signal, for example, adifference in reflective properties, the controller 156 can individuallyidentify and distinguish target sperm cells 160 from non-target spermcells 160. If the sperm cell 160 is a wanted sperm cell (i.e., one of X-or Y-chromosome sperm cell), a determination is made to allow the wantedor non-target sperm cell 160 to pass through the microfluidic channel164 to collection apparatus 188 unaffected. However, if the sperm cellis an unwanted sperm cell (i.e., the other of X- or Y-chromosome spermcell), the focused energy apparatus 157 will be employed to act upon thetarget sperm cell 160 after a predetermined delay time, to allow for theunwanted/target sperm cell to reach the action zone (which may be in thechamber 129, or between output 112 and collection apparatus.

The present invention allows for jitter in the system of the presentinvention, in order to have the most effective operation of the focusedenergy device 157 in the action zone, after the predetermined delaytime. Depending on the travelling velocity of the target sperm cell 160,the target sperm cell 160 will be in the action zone for about 2 .ts andthe focused energy beam 148 is most effective when aimed at the centerof the target sperm cell 160. Thus, it is preferable to limit jitter towithin 1 is or less.

As noted above, the target sperm cells 160 may be altered, photodamaged,killed, altered, disabled, or destroyed by either: 1) targeted or“pulse-on-demand” mode, or 2) a “continuous firing” mode, of the focusedenergy apparatus 157. As noted above, it is preferable to choose a laserwavelength for the focused energy device 157 that minimizes the requiredflux. For example, matching the laser wavelength to the absorbance ofthe dye used in the sperm cell staining can improve efficiency andeffectiveness. For example, if Hoechst 33342 dye is used for thestaining process, a 355 nm laser wavelength for focused energy apparatus157, is optimal.

In one embodiment, the target sperm cells are killed, or destroyed. Inanother embodiment, the target sperm cells 160 are sufficiently disabledsuch that they are no longer capable of performing a defined function.For example, a tail of the target sperm cell 160 may be disabled suchthat it no longer exhibits progressive motility. Thus, the target,disabled sperm cell 160 will be prevented from fertilizing an egg.

In one embodiment, sophisticated software for the controller 156 can bedesigned for a high power laser 157 meeting the requirements describedabove that allows single laser pulses 158 to be fired. Thus, thetargeted firing or “pulse-on-demand” mode delivers consistent laserpulses 158 whenever requested by the focused energy apparatus 157. Thetargeted firing mode is preferably used for samples 120 that containmostly non-target sperm cells 160, and only a relatively small number oftarget sperm cells 160 that need to be eliminated. However, with highspeed pulse-on-demand systems commercially available, the targetedfiring mode can be implemented for samples having other ratios ofnon-target to target sperm cells.

In an alternative embodiment, when target sperm cells 160 largelyoutnumber the non-target sperm cells 160, the focused energy apparatus157 may not be able to generate laser pulses 158 fast enough to act upon(i.e., kill or disable) all of the target sperm cells 160. Thus, thetargeted firing mode becomes less effective. In this case, the“continuous firing” mode becomes more favorable.

In the continuous firing mode, as discussed above, the focused energyapparatus 157 is a continuous wave (OW) or quasi-OW, or rapidly pulsedlaser 157, used to act on (i.e., kill or disable) every sperm cell 160passing through a certain location in the microfluidic channel 164without discriminating between target and non-target sperm cells 160.The focused energy beam 158 is shut off, deflected, or otherwiseinterrupted for a short period of time to allow non-target sperm cells(i.e., one of X-chromosome or Y-chromosome cells) to pass throughunaffected when a group of non-target sperm cells 160 is identified. Thegroup can be any predetermined number of non-target sperm cells 160.After the non-target sperm cells 160 pass the action zone, thecontinuous firing mode is re-started.

In both the targeted and continuous firing modes of operation, spermcells 160 that are too close to each other or that overlap one anotherin the sample fluid 120 stream are both killed, or both disabled, evenif one of the sperm cells 160 is a non-target sperm cell 160 instead ofa target sperm cell 160. As used herein, the term “too close” refers tothe presence of two or more sperm cells within range of the focusedenergy beam 158 such that both objects 160 are sufficiently acted uponby the focused energy beam 158 that the desired action occurs in bothcells. In addition, sperm cells 160 that are unable to be effectivelyidentified as either a non-target or target sperm cell 160, are killedor disabled by the focused energy beam 158, to ensure overall samplepurity for the discriminated product 165. Reasons that thediscriminating system of the present invention may be unable toeffectively identify a non-target sperm cell 160, may be due to flowissues within the microfluidic channel 164, staining problems, doublets,etc. Because the system of the present invention errs on the side ofkilling or disabling target sperm cells 160, or any sperm cells 160 thatcannot be effectively identified as either target or non-target, morepulses 158 may be used than the total number of actual target spermcells 160 present in the sample 129.

In one embodiment, as noted above, it is preferable to include a short“recharge/charge time” between laser pulses 158 of the focused energyapparatus 157. First, the spacing between two consecutive sperm cells160 is not uniform, but instead follows a Poisson distribution. In thesample fluid flow 120, there is a percentage of sperm cells 160 that arerelatively close to each other. To employ the focused energy apparatus157 at the events that have closer spacing (i.e., shorter elapsed time),less focused energy beam 158 “recharge/charge time” would be allowed.Further, if two sperm cells 160 are too close to each other such thattheir fluorescence signals interfere with each other, and no clearidentification of whether the sperm cell 160 is a target or non-targetcell, the focused energy apparatus 157 will have to fire multiple laserpulses 158 in a short period of time to kill or disable all of theunidentifiable sperm cells 160 in order to maximize sample purity. Thus,to achieve a higher throughput, generally a shorter average elapsed timebetween pulses 158 is required.

As noted above, the discriminated sample 120, after being acted upon bythe focused energy apparatus 157, is collected in a collection device188. Thus, in one embodiment, the collected product 165 contains boththe non-target sperm cells 160, and the target (i.e., killed, altered,destroyed, disabled) sperm cells 160, still in the same gender ratio asthe original sample 120. The collection in a single container 188 doesnot affect an overall sample 120 quality. Thus, in one embodiment, thefinal product 165 that is used for eventual fertilization includes boththe non-target and target sperm cells 160. Alternatively, subsequentproduct 165 separation techniques (i.e., flow cytometry, electrostaticplates, holographic optical trapping, etc.) may be used to, for example,separate the sperm cells 160 of the product 165 into live ordead/disabled sperm cells 160, or centrifugation may be used forremoving unwanted debris such as the remains of the killed or disabledtarget sperm cells 160.

In one embodiment, the microfluidic chip system of the present inventionis used in concert with a separation or isolation mechanism, such as theexemplary piezoelectric actuator assembly device described in pendingU.S. patent application Ser. No. 13/943,322, filed Jul. 16, 2013, or anoptical trapping system as described in in U.S. Pat. Nos. 7,241,988,7,402,131, 7,482,577, 7,545,491, 7,699,767, 8,158,927 and 8,653,442, forexample, the contents of all of which are herein incorporated byreference in their entirety.

In one embodiment, as shown in FIGS. 6B-C, the main channel 164 isshortened past the action chamber 129, such that the discriminatedobjects 160 leave the output channel 141 and output 112 in droplet 187form, before falling toward a collection apparatus 188. The operation ofthe focused energy apparatus 157 is the same, except that the objects160 are acted upon as they leave the exit of output channel 112 (seeFIG. 6B), or as the disconnected droplets 187 fall (see FIG. 60 ), andbefore they enter the collection apparatus 188.

Post-Collection of the Discriminated Product: In one embodiment, thediscriminated objects 160 are collected in a container 188 having 20%Tris (note: commercially available, such as sold by Chata Biosystems),which is disposed below the chip 100 output 112. In one embodiment, thecontents of the container 188 are circulated at predetermined intervals,to ensure mixing of the product 165 therein. In one embodiment, when thecontainer 188 reaches a predetermined volume (i.e., 18 ml), thecontainer 188 may be replaced with a new container 188.

In one embodiment, antibiotics are added to a filled container 188(i.e., 0.5 ml CSS antibiotics to 30 ml of product 165), the timerecorded, and a plurality of containers 188 with product 165 arecollected in a large container and cooled. In one example, threecontainers 188 with antibiotic-containing product 165, at a time, aredisposed in a 400 ml plastic container and placed in a cold room.

In one embodiment, after a predetermined amount of time in cooling(i.e., 2 hours). Tris B extender (14% glycerol Tris) is added in twoaliquots, a predetermined time apart (i.e., 15 minutes), to each tube.After the last sample 120 has cooled for a predetermined time (i.e., 2hours), and B extender is added, the samples 120 are centrifuged for 20minutes at 850 xG in a refrigerated centrifuge at 5° C. Supernatant isaspirated from each tube, leaving—0.2 ml with the pellet. All thepellets are combined into a single, pre-weighed container (i.e., tube),and a concentration of combined pellet using commercially availablecounting protocols (i.e., SpermVision) is determined, and the finalconcentration calculated to 11.35×106 cells/ml. The final volume isadjusted with a complete Tris A+B+CSS antibiotics extender (i.e., 50:50ratio of 20% egg yolk Tris+14% glycerol Tris).

In the exemplary embodiment of sperm cells 160, the printed semen strawsare filled and sealed, and the straws frozen using liquid nitrogenvapor.

In one embodiment, quality control measures post-freeze includebacterial contamination steps, where a frozen straw is thawed in a waterbath, and the thawed straw is wiped with alcohol to disinfect it. Thestraw contents are plated onto blood agar plate (5% sheep's blood), andincubated at 37° C. for 24 hours to determine the bacterial content.

In one embodiment, quality control measures for progressive motilityinclude thawing one frozen straw in a water bath, and expelling thestraw contents into a small tube placed in the tube warming stage.Commercial methods such as Motility Analysis or SpermVision are used todetermine the number of motile cells per straw.

In one embodiment, quality control measures for sample purity includethawing one frozen straw in a water bath, and isolating the live cellsby processing them through glass wool. Then FISH analysis is performedon the live cell population to determine the sex chromosome ratio.

Multiple Systems: In one embodiment, a plurality of microfluidic chips100, optical interrogation apparatuses and focused energy apparatuses,are set up in parallel to increase throughput.

Referring now to FIG. 18 , in one embodiment, a multiple systems layout400 with, for example, “n” systems 401, 402, etc., each equipped with afocused energy apparatus 157 is illustrated. In one embodiment, themultiple systems layout 400 includes a single interrogation apparatus147, and a single interrogation beam 148 which is configured to providea fluorescence excitation energy for the detection of objects 160—forexample, sperm DNA content.

In one embodiment, the multiple systems layout 400 further includes beamshaping optics 181, with a series of beam splitters 189, each beamsplitter providing a 50/50 split of the incoming power. This results innearly equal power beams 158 for each of the multiple systems 401, 402,etc.

External Devices

Separation Apparatus: In one embodiment, after the focused energyapparatus 157 acts on the objects 160 flowing through the microfluidicchip 100, instead of flowing into output channel 141, a separationmechanism (i.e., piezoelectric actuator assembly device (external orinternal), optical trapping assembly, electrostatic plates, or otherseparation mechanism well known to one of ordinary skill in the art, mayseparate the targeted and non-targeted objects 160. (See U.S. patentapplication Ser. No. 13/943,322, filed Jul. 16, 2013, or U.S. Pat. Nos.7,241,988, 7,402,131, 7,482,577, 7,545,491, 7,699,767, 8,158,927 and8,653,442, for example, the contents of all of which are hereinincorporated by reference in their entirety).

Thus, in one exemplary embodiment, targeted objects 160 that have beendamaged, killed, disabled, destroyed, or altered by the focused energyapparatus 157, can be separated from non-targeted objects 160, with theseparation mechanism separating the targeted objects into one of theoutput channels 140-142, and the non-targeted objects 160 into anotherof the output channels 140-142.

In another embodiment, the targeted objects are separated using aseparation mechanism after exiting from output 112, by use ofelectrostatic plates—well known in the art, for example.

In yet another embodiment, the focused energy apparatus 157 acts on theobjects 160 as they exit or drop from output 112, and the separationmechanism also separates the targeted objects 160 from the non-targetobjects 160 thereafter, or from product 165.

Pumping Mechanisms: As shown in FIGS. 19-21 , in one embodiment, apumping mechanism includes a system having a pressurized gas 235 whichprovides pressure for pumping sample fluid mixture 120 from reservoir233 (or sample tube 233), via tubing 242, into sample input 106 of themicrofluidic chip 100.

A central reservoir 240 (see FIGS. 20-21 ), or individual reservoirs 241(see FIG. 19 ), contain sheath or buffer fluids 163, and are connectedto each sheath or buffer input 107, 108, 172 of the microfluidic chip100 via tubing, in order to introduce sheath or buffer fluids 163therein. In one embodiment, the reservoirs are collapsible containers237 (see FIGS. 20-21 ), which are disposed in pressurized vessels 240,and the pressurized gas 235 pushes sheath or buffer fluids 163 to themicrofluidic chip 100. In one embodiment, the pressurized vessel 240pushes sheath or buffer fluids 163 to a manifold 238 (see FIG. 21 )having a plurality of different outputs, such that the sheath or bufferfluids 163 are delivered via tubing 231 a, 231 b, or 231 c to the sheathor buffer inputs 107, 108, or 172 respectively, of the chip 100.Although a three input sheath or buffer fluid arrangement is shown(i.e., FIG. 1D), one of ordinary skill in the art would know that lessor more tubing delivering sheath or buffer fluids to the microfluidicchip 100 is possible. In addition, although the tubing is shown asentering microfluidic chip 100 in FIGS. 18-21 , the disposition of thetubing with respect to the inputs on the chip 100 are not shown in exactorder. Further, one of ordinary skill in the art would know that thetubing enters the chip 100 via chip holder 200 (discussed below).

In one embodiment, each individual reservoir 241, or central reservoir240, includes a pressure regulator 234 which regulates the pressure ofthe gas 235 within the reservoir 241, 240 (see FIGS. 19-21 ). In oneembodiment, a pressure regulator 239 regulates the pressure of gas 235within the sample vessel 233. Flow meters 243 and flow valves 244control the sheath or buffer fluids 163 pumped via tubing 231 a, 231 b,or 231 c, respectively, into the sheath or buffer inputs 107, 108, or172, respectively (via holder 200). Thus, tubing 230, 231 a, 231 b, 231c, is used in the initial loading of the sheath or buffer fluids 120into the chip 100 and may be used throughout chip 100 operation to loadsample fluid 120 into sample input 106, or sheath or buffer inputs 107,108 (and 172).

In one embodiment, the flow meters 243 are used to provide the feedbackto flow valves 244 (see FIGS. 19-20 ) placed in the sheath flow routesto achieve a stable flow with constant flow rate in the microfluidicchannels. With the precise control of the flow, the overall flow ratevariation is less than 1% of the set flow rate, and the traveling speedto target objects 160 varies less than 1% during the detection andbetween interrogation/detection spot and action spot.

In one embodiment, sheath or buffer fluid 163 is pumped through a vacuumchamber (not shown) that is disposed in between the pressure canisterand the manifold, so as to remove dissolved gas in the sheath or bufferfluid. Inside the vacuum chamber, a segment of gas permeable tubing isdisposed between the input port and output port. While the sheath orbuffer fluid travels inside the vacuum chamber via the gas permeabletubing, the dissolved gas passes through the wall of the tubing, whilethe liquid remains inside the tubing. The applied vacuum helps gaspermeate through the tubing wall. In one embodiment, the gas-permeabletubing is made of a hydrophobic porous material, for example expandedpolytetrafluoroethylene (EPTFE), which has a high water entry pressurefor liquid but is permeable for gas.

Computer Control: In one embodiment, the user interface of the computersystem 156 includes a computer screen that displays the objects 160 in afield of view acquired by a CCD camera 182 over the microfluidic chip100.

In one embodiment, the computer 156 or a controller 156 controls anyexternal devices such as pumps (i.e., pumping mechanisms of FIGS. 19-20), if used, to pump any sample fluids 120, sheath or buffer fluids 163into the microfluidic chip 100, and also controls any heating deviceswhich set the temperature of the fluids 120, 163 being inputted into themicrofluidic chip 100.

In accordance with an illustrative embodiment, any of the operations,steps, control options, etc. may be implemented by instructions that arestored on a computer-readable medium such as a computer memory,database, etc. Upon execution of the instructions stored on thecomputer-readable medium, the instructions can cause a computing device156 to perform any of the operations, steps, control options, etc.described herein.

The operations described in this specification may be implemented asoperations performed by a data processing apparatus or processingcircuit on data stored on one or more computer-readable storage devicesor received from other sources. A computer program (also known as aprogram, software, software application, script, or code) can be writtenin any form of programming language, including compiled or interpretedlanguages, declarative or procedural languages, and it can be deployedin any form, including as a stand-alone program or as a module, object,subroutine, object, or other unit suitable for use in a computingenvironment. A computer program may, but need not, correspond to a filein a file system. A program can be stored in a portion of a file thatholds other programs or data (e.g., one or more scripts stored in amarkup language document), in a single file dedicated to the program inquestion, or in multiple coordinated files (e.g., files that store oneor more modules, sub-programs, or portions of code). A computer programcan be deployed to be executed on one computer or on multiple computersthat are located at one site or distributed across multiple sites andinterconnected by a communication network. Processing circuits suitablefor the execution of a computer program include, by way of example, bothgeneral and special purpose microprocessors, and any one or moreprocessors of any kind of digital computer.

Microfluidic Chip Holder: In one embodiment, the microfluidic chip 100is loaded on a chip holder 200 (see FIGS. 22A-23B). The chip holder 200is mounted to a translation stage (not shown) to allow positioning ofthe holder 200 with respect to the interrogation apparatus and focusedenergy apparatus. The microfluidic chip holder 200 is configured to holdthe microfluidic chip 100 in a position such that the light beam 148from the interrogation apparatus may intercept the objects 160 in theabove described manner, at opening 150, and the focused energy apparatus157 may act upon the objects 160.

The mechanisms for attachment of the chip 100 to the holder 200 aredescribed below along with their method of operation, but one ofordinary skill in the art would know that these devices may be of anyconfiguration to accommodate the microfluidic chip 100, as long as theobjectives of the present invention are met.

As illustrated in FIGS. 22A-23B, in one embodiment, a microfluidic chipholder 200 is made of a suitable material, such as aluminum alloy, orother suitable metallic/polymer material, and includes main plate 201and fitting plate 202. The main plate 201 and fitting plate 202 may beof any suitable shape, but their configuration depends on the layout ofthe chip 100 and the requirements for access thereto.

In one embodiment, the main plate 201 has a substantial L-shape, but theshape of the holder 200 depends on the layout of the chip 100 (see FIGS.22A-23B). One leg of the L-shaped main plate 201 includes a plurality ofslots that are configured to receive mounting screws 219, which are usedto mount the holder 200 on the translation stage. Any number of slots ofany suitable shape and size, may be included in the main plate 201. Inone embodiment, four screws 219 are used to mount the holder 200 on thetranslation stage and adjust the position of the holder 200. However,one of ordinary skill in the art would know that any number of any sizeslots and mounting screws may be used.

In one embodiment, a switch valve 220 is attached to the main plate 201above the mounting screws 219 on the one leg of the L-shaped main plate201 (see FIGS. 22A-23B). In addition, in the other leg of the L-shapedmain plate 201, a pair of linear actuators, such as pneumatic cylinderactuators 207A and 207B, are disposed on the same side of the main plate201 as the switch valve 220 (see FIGS. 22A-23B).

In one embodiment, a fitting plate 202 is attached to the main plate201, on the other side and along the other leg of the L-shaped mainplate 201 (see FIGS. 22A-23B). The fitting plate 202 includes aplurality of apertures that accommodate a plurality of fittings 204-206(see FIGS. 22A-22B, for example) configured to receive and engage withexternal tubing (see FIGS. 19-23B) for communicating fluids/samples tothe microfluidic chip 100. In one embodiment, the fitting plate 202includes three fittings 204-206, which are used to align with the sheathor buffer inputs 107, 108, and sample input 106, respectively (see FIGS.1B and 22A-22B). In another embodiment, four fittings 204-206 and 216,are used to align with the sheath or buffer inputs 107, 108, sampleinput 106, and sheath or buffer input 172, respectively (see FIGS. 1Aand 23A-23B). However, one of ordinary skill in the art would know thatthe fittings would be arranged in any way by number and position, so asto align with the number of inputs in the microfluidic chip 100 andallow transmission of fluids from one or more reservoirs via tubing (seeFIGS. 19-21 ).

In one embodiment, a pair of slot washers 203A, 203B affix the fittingplate 202 and pneumatic cylinder actuators 207A, 207B to the main plate201 (see FIGS. 22A-23B). Thus, the fitting plate 202 is easily removedfrom the main plate 201 without disassembling the switch valve 220 andpneumatic cylinder actuators 207A, 207B from the main plate 202.

In one embodiment, the pneumatic cylinder actuators 207A, 207B eachinclude a piston (not shown) having a piston rod coupled thereto andextendable and retractable relative to the cylindrical body portion. Inone embodiment, air is supplied through port 209 into the switch valve208, and exits through ports 210, 211 to ports 212, 213, respectively,in pneumatic cylinder actuator 207A. The air is further supplied fromports 214A, 214B in pneumatic cylinder actuator 207A, to ports 215A,215B, respectively, in pneumatic cylinder actuator 208A.

The toggle switch 220 of switch valve 208 opens and closes, to allow orprevent, respectively, the air from the air supply entering through port209 and to pneumatic cylinder actuators 207A, 207B. When the air isprovided to the pneumatic cylinder actuators 207A, 207B, the piston rodsof the pneumatic cylinder actuators 207A, 2073 are extended outwardly toan open position (not illustrated), and fitting plate 202 is pushedoutwardly away from the microfluidic chip 100 to an open position toallow a user to load (or unload) the microfluidic chip 100 (see FIGS.22A-23B). When the air supply is closed, the piston rods of thepneumatic cylinder actuators 207A, 207B are retracted inwardly to aclosed position (as illustrated in FIGS. 22A-23B), and the fitting plate202 is drawn towards the chip 100 and pressed against the chip 100forming a liquid seal between the chip 100 and the connections to thetubing for the sheath or buffer and sample fluids.

In one embodiment, when the microfluidic chip 100 is in the closedposition, O-rings, which are disposed on the surface of the fittingplate 202, between the fitting plate 202 and the chip 110, form asubstantially leak-free seal to protect the microfluidic chip 100 fromdamage. However, one of ordinary skill in the art would know that anynumber and configuration of O-rings or gaskets may be used.

In one embodiment, the holder 200 is positioned such that the chip 100is at a sufficient height to accommodate at least one collection vessel188 disposed underneath the chip 100. In another embodiment, collectionvessels are disposed between each output 111-113.

In another embodiment, the present invention discloses a method ofinseminating an animal comprising flowing a stream of a population ofsperm cells through a channel, differentiating the sperm cells into atleast two subpopulations with one subpopulation comprising X-chromosomecontaining sperm cells and another subpopulation comprising Y-chromosomecontaining sperm cells, selecting a desired subpopulation, ablating anundesired subpopulation, and collecting both the desired subpopulationand the ablated undesired subpopulation together, wherein the collectedpopulation of sperm cells is used to fertilize an egg. Insemination isthe deliberate introduction of sperm into a female animal for thepurpose of impregnating or fertilizing the female for sexualreproduction. Insemination is inclusive of introduction of a fertilizedegg into a female animal. X chromosome and Y chromosome are both sexchromosomes, and generally, two X chromosomes are present in femalecells, and generally, only one X chromosome and one Y chromosome arepresent in male cells. Fertilization is defined as the fusion of maleand female gametes to form a zygote, a fertilized egg. Specifically, asperm cell unites with an egg. Differentiating sperm cells includesidentifying a physical or a genetic characteristic of a sperm cellincluding, but not limited to, whether a sperm cell has an X-chromosomeor a Y-chromosome. Selecting a desired subpopulation includes targetinga subpopulation of cells for a certain action, for instance, ablatingthe subpopulation of cells while not ablating a different subpopulationof cells. Ablating an undesired subpopulation is defined as renderingsperm cells deficient in fertilizing an egg. It could be further definedas rendering sperm cells incapable of fertilizing an egg. Collectingsubpopulations of sperm cells includes gathering both the selected andthe unselected subpopulations together, usually in a container, a tubeor a straw. It could be further defined as the stream of population ofsperm cells are not physically subdivided prior to and subsequent toablation of a subpopulation of sperm cells and that both subpopulationsexit together and are gathered.

In a further embodiment, the present invention includes inserting thecollected population of sperm cells into the mammal (human or animal) tofertilize an egg. Given that sperm cells outside the body are generallysubject to conditions that are unfavorable for their survivability,fertilization should occur as soon as possible following collection. Insome embodiments, the insertion occurs within forty-eight hoursfollowing the collection.

In a further embodiment, the present invention includes fertilizing theegg in vitro with the collected population of sperm cells to create afertilized egg and implanting the fertilized egg within the animalwherein the implantation occurs within forty-eight hours followingfertilization. In vitro fertilization is a process of creating embryosfrom oocytes (unfertilized egg cells) by fertilizing them with semenoutside the uterus. Implantation of the fertilized egg within the mammal(human or animal) includes, at least, the placement of the fertilizedegg in any part of the female mammal (human or animal), such as theuterus to allow for the development of the fertilized egg into an embryoaccording to the genetics of the fertilized egg.

In a further embodiment, the present invention includes obtaininggenetic information of the sperm cells or the fertilized egg prior toimplantation. In some embodiments, at least one, alternatively at leasttwo cells of the fertilized egg for analysis. In other embodiments, aportion of a DNA molecule or an RNA molecule of the fertilized egg issequenced. In some embodiments, yj DNA or RNA sequencing occurs withinforty-eight hours following removal of at least one, alternatively atleast two cells of the fertilized egg for analysis. Alternatively, theDNA or RNA sequencing occurs within twenty-four hours following removalof at least one, alternatively at least two cells of the fertilized eggfor analysis. Obtaining genetic information, i.e., DNA or RNAsequencing, of the sperm cells or the fertilized eggs includes assessingthe genetic profile or a gene or a set of genes to determine the qualityof the cells and/or the phenotypic trait(s) of the grown animal.Alternatively, probes, including oligonucleotides could be used toascertain the genetic sequence or profile of certain genes. DNA or RNAarrays could be used to assess the genetic profile of a cell. All knownmolecular biological and cellular techniques could be employed to assessthe characteristics and the identities of the cells and their likelyphenotypic outcome as an animal. Such analyses typically occur within ashort period such as within forty-eight hours, alternatively withintwenty-four hours to lessen any damage to lessen any damage to the spermcells or the fertilized eggs.

In a further embodiment, implanting the fertilized egg within the mammal(human or animal) occurs within forty-eight hours following obtainingthe genetic information of the fertilized egg. The sooner a fertilizedegg is implanted within a mammal (human or animal), the better to ensuresurvivability.

With respect to flowing the stream of the population of sperm cellsthrough the channel, a further embodiment includes orienting the spermcells in the population in a particular position with respect to thechannel, aligning the sperm cells wherein the sperm cells are flowingsingularly through the channel, and exiting all the sperm cells throughan output portion of the channel. With respect to orienting the spermcells and aligning the sperm cells, some embodiments include compressingthe stream by sheath fluid flowing along at least one side of thestream, and wherein the compressing further comprises intersecting at asubstantially 90 degree vertical plane by the sheath fluid from abovethe stream. In other embodiments, the sheath fluid is in laminar flowwith the stream of the population of sperm cells during thedifferentiating and the ablating steps. In an alternative embodiment,orienting the sperm cells and aligning the sperm cells includescompressing the sperm cells by an inertial force directed by a designedstructure of the channel. Orienting includes placing the cell in aproper three-dimensional space within the channel to allow for properdetermination of characteristics of the cells. Aligning includes placingthe cells in proper order within the channel, and in most cases, theproper order is cells in a single file line with consistent orientation.Exiting all the sperm cells through an output portion of the channelincludes that the selected and unselected sperms are not separated fromeach other or their path diverged to a separate path or channel duringcollection.

In another embodiment, differentiating the sperm cells into twosubpopulations of X-chromosome containing sperm cells and Y-chromosomecontaining sperm cells includes illuminating the sperm cells with a beamof light, and detecting a difference between the two subpopulations ofsperm cells. In a further embodiment, the beam of light is from adetection laser. FIG. 27 shows a multiple station setup to analyze cellswith a vanguard laser. In another embodiment, the beam from the laser issplit into multiple beams, and each beam of the multiple beams isconfigured to illuminate a different sperm cell. In another embodiment,the beam from the laser is not split into multiple beams and the beamilluminates one sperm cell at a time. FIG. 28 shows beam splittingoccurring for multiple stations. The beam from the laser is emitted as acontinuous wave, or it is emitted in pulses. In an alternativeembodiment, the beam of light is from an LED light source.

In another embodiment, the difference comprises how the sperm cellsreflect, refract, diffract, absorb, and/or emit the light. In someinstances, a molecule of the sperm cell or a molecule associated withthe sperm cell is excited by the beam and the molecule emits a secondbeam in a Stoke shift. FIG. 29 shows that the Stoke shift creates a peakat 450 nm wavelength, therefore, creating the detection wavelength. Thedifference reflects a difference in DNA concentration per cell. As seenin FIG. 24 , the difference between the subpopulations is at least about3.8% difference in DNA content. The approximate 3.8% difference reflectsthe difference between sperm cells with the X-chromosome compared withsperm cells with the Y-chromosome. For bovines, the female sperm cellhas the X-chromosome and 29 autosomes and male sperm cell has theY-chromosome and 29 autosomes. FIG. 25 shows staining of DNA in cells todetermine the intensity of emitted light which correlates with theamount of DNA in a cell, wherein 3.8% more light emitted correlates to3.8% more DNA in female sperm cells.

In another embodiment, selecting the desired subpopulation of particlesincludes gating each subpopulation based on a physical difference foreach subpopulation, and identifying the desired subpopulation. Gatingeach subpopulation and identifying the desired subpopulation includescorrelating a graphic representation for each subpopulation and choosingor marking the desired subpopulation. As the cells travel through thechannel, they are graphically captured and their particularcharacteristic(s) are plotted on a graph. With each cell plotted on agraph, a gating of any particular population is possible wherein aparticular subpopulation is selected or designated for certain action,such as laser ablation rendering certain sperm cells incapable offertilizing an egg.

In another embodiment, ablating the undesired subpopulation furtherincludes damaging or killing the undesired subpopulation of sperm cellswith a second beam of light in an area of the channel, wherein a damagedor killed sperm cell is rendered unable to fertilize an egg. In someembodiments, the second beam of light is from a laser. In a furtherembodiment, the laser is configured to provide a pulse of beam. In yetanother embodiment, the laser is configured to recharge to damage orkill the next sperm cell that enters the area of the channel. As seen inFIGS. 30A and 30B in some embodiments, the beam of light is tightlyfocused with a blade-shape configured to cut across approximately 2microns in length of the channel to damage or kill the cell. As seen inFIG. 30B, there is an initial detection of the cell, which triggers apulse laser based on a timer, and knowing the distance of travel (e.g.,175 microns), the pulse laser damages or kills the cell. In otherembodiments, the damaging or killing is coordinated with the travel timeof the sperm cells and wherein the sperm cells are traveling at a knownvelocity. The damaging or killing is coordinated so that the laser hitsany particular cell as it travels through the channel. The coordinationis timed so that only certain cells are hit with the laser beam.

In another embodiment, the animal is a cow or a heifer. A heifer is ayoung female cow that has not borne a calf. A cow is one that haspreviously borne a calf. In a further embodiment, the success rate forinsemination of the heifer is at least about 60% on average. In analternative embodiment, the success rate for insemination of the cow isat least about 37% on average. The success rate for insemination of theheifer is better compared with insemination comprising a sexing processthat comprises a physical separation of selected sperm cells fromunselected sperm cells. Table 2 depicts conception rates (CR) of cattleherds that have received different kinds of insemination technology:sexing technology of the present invention (IntelliGen SexedTechnology), conventional insemination without sexing, and competitors'sexing technology. Different types of heifers (Holstein and Jersey) wereinseminated. Over 6000 recorded insemination were analyzed. IntelliGenSexed Technology achieved 63.8% conception rate for Holstein heifers and61.9% conception rate for Jersey heifers. For Holstein (HO) heifers, theCR (61.9%) of insemination using IntelliGen Sexed Technology iscomparable to the CR (64.6%) for heifer's that received insemination ofunsexed sperms (conventional) and much better to the CR (54.7%) ofinsemination from competitors' sexing technology, suggesting thatIntelliGen Sexed Technology is less detrimental compared withcompetitors' sexing processes. Competitors' sexing processes comprise aphysical separation of selected sperm cells from unselected sperm cells.In contrast, IntelliGen Sexed Technology does not require physicalseparation. Both selected and unselected sperm cells are collectedtogether—only that the undesired subpopulation of sperm cells has beenrendered incapable of fertilization. For Jersey (JE) heifers, the CR(61.9%) of insemination from IntelliGen Sexed Technology wassurprisingly much better compared with the CR (38%) of conventionalinsemination as well as the CR (54.7%) of insemination from competitors'sexing technology. Table 3 depicts a comparison of three types ofinsemination of cows are analyzed with respect to CR: IntelliGen SexedTechnology (42.6% for HO; 37.5% for JE); unsexed conventionalinsemination (40.3% for HO; 44.0% for JE), and competitors' sexingtechnology (36.3% for HO; 34.2% for JE) wherein the desired sperm cellsare physically separated from the undesired sperm cells. Consistently,the CR using the IntelliGen Sexed Technology was comparable to the CRusing conventional insemination technique while they were both bettercompared to the CR using competitors' sexing technology.

TABLE 2 Heifer Insemination Records on Farms that used all 3 technologytypes (conventional, Genus Sexed Semen (IntelliGen), Other SexedTechnology): IntelliGen Other Sexed Tech Conventional Sexed Tech.Heifers (N) CR % (N) CR % (N) CR % Farms (N) HO (1109) 63.8% (398) 64.6(1785) 54.7% 14 JE  (155) 61.9%  (50) 38%  (245) 47.8% 1

TABLE 3 Cow Insemination records on farms that used all 3 technologytypes. The inseminations were from September 1 and only on 1st and 2ndservice on cows: IntelliGen Other Sexed Tech Conventional Sexed Tech.Cows (N) CR % (N) CR % (N) CR % Farms (N) HO  (665) 42.6% (3510) 40.3% (259) 36.3% 7 JE (2762) 37.5% (1037) 44.0% (2794) 34.2% 5

In another embodiment; the current invention discloses a collection of apopulation of sperm cells with both a desired subpopulation and anablated undesired subpopulation according to the method of inseminatingan animal that comprises flowing a stream of a population of sperm cellsthrough a channel, differentiating the sperm cells into twosubpopulations of X-chromosome containing sperm cells and Y-chromosomecontaining sperm cells, selecting a desired subpopulation, ablating anundesired subpopulation, and collecting both the subpopulations of spermcells including the desired subpopulation and the ablated undesiredsubpopulation together, wherein the collected population of sperm cellsis used to fertilize an egg.

In another embodiment, the current invention discloses an embryo,wherein the embryo is made from the step of inserting the collectedpopulation of sperm cells into the animal to fertilize an egg. In analternative embodiment, the embryo is made from fertilizing the egg invitro with the collected population of sperm cells creating a fertilizedegg, and implanting the fertilized egg within the mammal (human oranimal), and wherein the implantation occurs within forty-eight hoursfollowing fertilization.

Further aspects and embodiments of the present technology are describedin the following paragraphs.

A method of producing a sperm cell composition the steps of: flowing astream of a population of sperm cells through a channel; differentiatingthe sperm cells into two subpopulations of X-chromosome containing spermcells and Y-chromosome containing sperm cells; selecting a desiredsubpopulation; ablating an undesired subpopulation; and collecting boththe subpopulations of sperm cells including the desired subpopulationand the ablated undesired subpopulation together.

A method of fertilizing one or more eggs comprising the steps ofproviding at least one egg obtained from a female mammal, providing thesperm cell composition from a male mammal of the same species producedusing the method described above as the female mammal, and mixing theone or more eggs with the sperm cell composition. An aspect of themethods described above, wherein the female mammal is a cow or a heifer.An aspect of the methods described above, wherein the male mammal is abull or boar. An aspect of the methods described above, furthercomprising implanting the at least one fertilized egg within the femaleanimal within forty-eight hours following fertilization. An aspect ofthe methods described above, further comprising obtaining geneticinformation of the sperm cells or the fertilized egg prior toimplantation. An aspect of the methods described above, whereinimplanting the fertilized egg within the female animal occurs withinforty-eight hours following obtaining the genetic information of thefertilized egg. An aspect of the methods described above, whereinobtaining the genetic information of the fertilized egg comprisesremoving at least one cell of the fertilized egg for analysis. An aspectof the methods described above, wherein obtaining the geneticinformation of the fertilized egg comprises sequencing a portion of aDNA molecule or an RNA molecule of the fertilized egg. An aspect of themethods described above, wherein obtaining the genetic information ofthe fertilized egg occurs within forty-eight hours following removing atleast one cell of the fertilized egg for analysis. An aspect of themethods described above, wherein obtaining the genetic information ofthe fertilized egg occurs within twenty-four hours following removing atleast one cell of the fertilized egg for analysis.

A method of producing an embryo comprising using the sperm cellcomposition produced using the method described above and an assistedreproductive technique. An aspect of the methods described above,wherein the assisted reproductive technique is selected from the groupconsisting of in vitro fertilization (IVF), artificial insemination(AI), intracytoplasmic sperm injection (ICSI), multiple ovulation andembryo transfer (MOST), and other embryo transfer techniques. An aspectof the methods described above, wherein a heifer or cow is inseminatedusing artificial insemination. An aspect of the methods described above,wherein success rate for insemination is better than inseminationcomprising a physical separation of selected sperm cells from unselectedsperm cells. An aspect of the methods described above, wherein successrate for insemination of a heifer is at least about 60% on average. Anaspect of the methods described above, wherein success rate forinsemination of the cow is at least about 37% on average.

An aspect of the methods described above, wherein flowing the stream ofthe population of sperm cells through the channel comprises orientingthe sperm cells in the population in a particular position with respectto the channel; aligning the sperm cells wherein the sperm cells areflowing singularly through the channel; and exiting all the sperm cellsthrough an output portion of the channel. An aspect of the methodsdescribed above, wherein orienting the sperm cells and aligning thesperm cells comprise compressing the stream by sheath fluid flowingalong at least one side of the stream wherein the compressing furthercomprises intersecting at a substantially 90-degree vertical plane bythe sheath fluid from above the stream. An aspect of the methodsdescribed above, wherein the sheath fluid is in laminar flow with thestream of the population of sperm cells during the differentiating andthe ablating steps. An aspect of the methods described above, whereinorienting the sperm cells and aligning the sperm cells comprisescompressing the sperm cells by an inertial force directed by a designedstructure of the channel. An aspect of the methods described above,wherein differentiating the sperm cells into two subpopulations ofX-chromosome containing sperm cells and Y-chromosome containing spermcells is accomplished via the steps of: illuminating the sperm cellswith a beam of light; and detecting a difference between the desiredsubpopulation and the ablated undesired subpopulation. An aspect of themethods described above, wherein the beam of light is from an LED lightsource. An aspect of the methods described above; wherein the beam oflight is from a detection laser. An aspect of the methods describedabove, wherein the beam of light is split into multiple beams of light;and wherein each beam of light of the multiple beams of light isconfigured to illuminate a different sperm cell. An aspect of themethods described above, wherein the beam of light is not split intomultiple beams and the beam illuminates one sperm cell at a time. Anaspect of the methods described above, wherein the beam of light isemitted as a continuous wave. An aspect of the methods described above;wherein the beam of light is emitted in pulses. An aspect of the methodsdescribed above, wherein the difference comprises how the sperm cellsreflect, refract, diffract, absorb, and/or emit the light. An aspect ofthe methods described above, wherein a molecule of the sperm cell or amolecule associated with the sperm cell is excited by the beam of lightand the molecule emits a second beam of light in a Stoke shift. Anaspect of the methods described above, wherein the difference reflects adifference in DNA concentration per cell. An aspect of the methodsdescribed above, wherein the difference between the subpopulations is atleast about 3.8% difference in DNA content.

An aspect of the methods described above, wherein selecting the desiredsubpopulation comprises gating each subpopulation based on a physicaldifference for each subpopulation; and identifying the desiredsubpopulation. An aspect of the methods described above, wherein gatingeach subpopulation and identifying the desired subpopulation comprisescorrelating a graphic representation for each subpopulation; andchoosing or marking the desired subpopulation. An aspect of the methodsdescribed above, wherein ablating the undesired subpopulation furthercomprises damaging or killing the undesired subpopulation of sperm cellswith a second beam of light in an area of the channel, wherein a damagedor killed sperm cell is rendered unable to fertilize an egg. An aspectof the methods described above, wherein the second beam of light is froma laser. An aspect of the methods described above, wherein the laser isconfigured to provide a pulse of the beam of light and wherein the laseris configured to recharge to damage or kill the next sperm cell thatenters the area of the channel. An aspect of the methods describedabove, wherein the beam of light is tightly focused with a blade shapeconfigured to cut across at least approximately 2 microns in length ofthe channel. An aspect of the methods described above, wherein thedamaging or killing is coordinated with travel time of the sperm cellsand wherein the sperm cells are traveling at a known velocity.

A process for the production of at least one fertilized egg from afemale mammal, the process comprising the steps of a) flowing a streamof a population of sperm cells through a channel; b) differentiating thesperm cells into two subpopulations of X-chromosome containing spermcells and Y-chromosome containing sperm cells; c) selecting a desiredsubpopulation; d) ablating an undesired subpopulation; e) collectingboth the subpopulations of sperm cells including the desiredsubpopulation and the ablated undesired subpopulation together; and f)mixing at least a portion of the collected sperm cells with at least oneegg from a female mammal to produce a fertilized egg. An aspect of theprocess described above, further comprising implanting the at least onefertilized egg within the female animal within forty-eight hoursfollowing fertilization. An aspect of the process described above,further comprising obtaining genetic information of the sperm cells orthe fertilized egg prior to implantation.

A process for the production of at least one embryo from a femalemammal, the process comprising the steps of a) flowing a stream of apopulation of sperm cells through a channel; b) differentiating thesperm cells into two subpopulations of X-chromosome containing spermcells and Y-chromosome containing sperm cells; c) selecting a desiredsubpopulation; d) ablating an undesired subpopulation; e) collectingboth the subpopulations of sperm cells including the desiredsubpopulation and the ablated undesired subpopulation together; f)inserting at least a portion of the collected sperm cells into a femalemammal; and g) fertilizing at least one egg from said female mammal toproduce at least one embryo from said female mammal.

The construction and arrangements of the microfluidic chip system with afocused energy device, as shown in the various illustrative embodiments,are illustrative only. It should be noted that the orientation ofvarious elements may differ according to other illustrative embodiments,and that such variations are intended to be encompassed by the presentdisclosure. Although only a few embodiments have been described indetail in this disclosure, many modifications are possible (e.g.,variations in sizes, dimensions, structures, shapes and proportions ofthe various elements, values of parameters, mounting arrangements, useof materials, colors, orientations, etc.) without materially departingfrom the novel teachings and advantages of the subject matter describedherein. Some elements shown as integrally formed may be constructed ofmultiple parts or elements, the position of elements may be reversed orotherwise varied, and the nature or number of discrete elements orpositions may be altered or varied. The order or sequence of anyprocess, logical algorithm, or method steps may be varied orre-sequenced according to alternative embodiments. Other substitutions,modifications, changes and omissions may also be made in the design,operating conditions and arrangement of the various illustrativeembodiments without departing from the scope of the present disclosure.All such modifications and variations are intended to be included hereinwithin the scope of the invention and protected by the following claims.

What is claimed is:
 1. A flow cytometry apparatus configured to producea sexed composition of sperm comprising non-human animal sperm byidentifying a subpopulation of sperm from a population of sperm in asample fluid, the flow cytometry apparatus comprising: an interrogationlight source; a detector; and a computer readable media, the computerreadable media comprising instructions that when executed by aprocessor; define and store a gating strategy for differentiating thesubpopulation of sperm in the sample fluid in the population of spermbased on one or more physical characteristics of sperm in the populationof sperm, wherein the one or more physical characteristics areidentifiable within the population of sperm based in part oninterrogation by the interrogation light source as detected by thedetector, and wherein the gating strategy comprises identifying one ormore desired physical characteristics of the subpopulation of sperm;measure the one or more physical characteristics for the sperm in thepopulation of sperm based on a set of signals detected by the detector;update the gating strategy based on the one or more physicalcharacteristics measured for the sperm in the population of sperm; storethe updated gating strategy; and identify sperm in the subpopulation ofsperm based on the measured one or more physical characteristics and theupdated gating strategy.
 2. The flow cytometry apparatus of claim 1,wherein the computer readable media further comprises instructions togenerate the sexed composition of sperm comprising the subpopulation ofsperm identified based on the measured one or more physicalcharacteristics and the updated gating strategy.
 3. The flow cytometryapparatus of claim 1, wherein the computer readable media furthercomprises instructions to set a timing setting based on the one or morephysical characteristics measured for the sperm in the population ofsperm.
 4. The flow cytometry apparatus of claim 1, wherein the timingsetting provides for jitter.
 5. The flow cytometry apparatus of claim 1,wherein the computer readable media further comprises instructions tomeasure a span distribution.
 6. The flow cytometry apparatus of claim 5,wherein the updated gating strategy is updated based in part on the spandistribution.
 7. The flow cytometry apparatus of claim 1, wherein thepopulation of sperm in the sample fluid comprises bovine sperm.
 8. Theflow cytometry apparatus of claim 1, wherein the sexed composition ofsperm substantially comprises X chromosome-bearing sperm cells.
 9. Theflow cytometry apparatus of claim 1, wherein the sexed composition ofsperm substantially comprises Y chromosome bearing sperm cells.
 10. Theflow cytometry apparatus of claim 1, wherein a physical characteristicof the one or more physical characteristics is an amount of DNA.
 11. Theflow cytometry apparatus of claim 1, wherein the computer readable mediafurther comprises instructions to perform an action on each sperm cellin a set of unidentifiable sperm cells, wherein the unidentifiable spermcells are not able to be identified based on the updated gatingstrategy.
 12. The flow cytometry apparatus of claim 1, wherein thecomputer readable media further comprises instructions to perform anaction on each sperm cell in a set of too close sperm cells.
 13. Anon-transitory computer readable media for use with a flow cytometryinstrument, the computer readable media comprising instructions thatwhen executed by a processor cause the flow cytometry instrument todefine and implement a gating strategy for use in producing a sexedcomposition of sperm comprising non-human animal sperm by identifying asubpopulation of sperm from a population of sperm in a sample fluid, theinstructions comprising: measure one or more physical or geneticcharacteristics present in the sexed composition of sperm based on anoutput of a detector; set gating parameters for differentiating thesubpopulation of sperm in the sample fluid in the population of spermbased on the one or more physical or genetic characteristics of spermmeasured in the population of sperm resulting in a defined gatingstrategy, wherein the gating parameters comprise marking one or moredesired physical or genetic characteristics of the subpopulation ofsperm; store the defined gating strategy; measure the one or morephysical or genetic characteristics for the sperm in the population ofsperm based on the defined gating strategy; update the defined gatingstrategy based on the measured one or more physical or geneticcharacteristics for the sperm to generate an updated gating strategy;store the updated gating strategy; identify sperm in the subpopulationof sperm based on the measured one or more physical or geneticcharacteristics and the updated gating strategy.
 14. The non-transitorycomputer readable media of claim 13, wherein instructions furthercomprise instructions to: generate the sexed composition of spermcomprising the subpopulation of sperm identified based on the measuredone or more physical or genetic characteristics and the updated gatingstrategy.
 15. The non-transitory computer readable media of claim 13,wherein instructions further comprise instructions to: set a timingsetting based on the one or more physical or genetic characteristicsmeasured for the sperm in the population of sperm.
 16. Thenon-transitory computer readable media of claim 15, wherein the timingsetting provides for jitter.
 17. The non-transitory computer readablemedia of claim 13, wherein instructions further comprise instructionsto: measure a span distribution.
 18. The non-transitory computerreadable media of claim 17, wherein the updated gating strategy isupdated based in part on the span distribution.
 19. The non-transitorycomputer readable media of claim 13, wherein the population of sperm inthe sample fluid comprises bovine sperm.
 20. The non-transitory computerreadable media of claim 13, wherein the sexed composition of spermsubstantially comprises X chromosome-bearing sperm cells.
 21. Thenon-transitory computer readable media of claim 13, wherein the sexedcomposition of sperm substantially comprises Y chromosome bearing spermcells.
 22. The non-transitory computer readable media of claim 13,wherein a physical or genetic characteristic of the one or more physicalor genetic characteristics is an amount of DNA.
 23. The non-transitorycomputer readable media of claim 13, wherein instructions furthercomprise instructions to: perform an action on each sperm cell in a setof unidentifiable sperm cells, wherein the unidentifiable sperm cellsare not able to be identified based on the updated gating strategy. 24.The non-transitory computer readable media of claim 13, whereininstructions further comprise instructions to: perform an action on eachsperm cell in a set of too close sperm cells.